Portal - Did You Know Gaming? Feat. MatPat from Game Theory
There are unused lines for Cave Johnson found within the games '.nut' files. The lines would've been used for a scene where the player finds an AI cube with Cave Johnson's conscious programmed inside it. He would meet GLaDOS and reveal that she indeed used to be Caroline and that he "volunteered" her to be a test subject to see if his conscious could be loaded into a machine. He would then continuously ask the player to kill him.
Contributed by ShyanVixen
There was originally going to be a song featured in the game called "GLaDOS' Song". It was written and performed by Ellen McLain, the voice actor for GLaDOS. She wrote the song for use as an Easter Egg in the game but it was ultimately never used.

The song was actually recorded after the game's release, and was performed by both Ellen and her husband, John Lowrie, the voice of the Sniper from Team Fortress 2. It was later modified by Harry Callaghan to sound closer to GLaDOS' synthesized voice.
Contributed by Arachnus
After the Authoring Tools update, the Adhesion Gel (which allowed Chell to defy gravity) was replaced with the Reflection Gel, a gel that reflects lasers. It also reflects the turrets' laser sights, but this is just a cosmetic feature and doesn't affect their actual "vision".
Contributed by PoisonTardis
There is an unused Turret hidden in the game files called the Hover Turret. It has no programming other than to follow the player with its laser sight. It also has no model, so it shows up as a default ERROR model.
Contributed by PoisonTardis
In an earlier version of the co-op plot, GLaDOS sends Atlas and P-Body out to find "artifacts" of the long-dead Aperture workers (coffee cups and such) to make them more human. Unused audio files from this period of development can still be found within the games files, revealing that in one of the rooms a comic strip is found, presumably Garfield from how it is described.
Contributed by Boyobmas
During the development of Portal 2, playtesters found GLaDOS to be far too mean and vindictive. The developers concluded that a shift in personality was necessary, to better reflect the change in the power dynamic between Chell and GLaDOS. As a result, some of her dialogue was changed to make GLaDOS more amicable and passive-aggressive.
Contributed by RetroRex
When Portal 2's audio files are tampered with or deleted, in the place of a broken audio clip, the game will instead play a file called "error.wav". This audio clip is of Dr. Kleiner from Half-Life 2, taken from a scene in which Dr. Kleiner is beginning to teleport Alyx but the cord gets unplugged, saying "Oh fiddlesticks, what now?"
Contributed by ILoveMaMum
During Chapter 2, it is possible to grab Wheatley as he comes out of the wall in Test Chambers 2 and 7. He cannot be harmed by lasers and will not be destroyed by the Emancipation Grill, but disappears after taking the elevator to the next Test Chamber.
Contributed by Pogue-Mahone
The paintings in Chell's room at the beginning of the game changes from a day scene to a night scene depending on whether it is daytime or nighttime.
Contributed by Psychospacecow
There is an achievement/trophy in the game called Ship Overboard. In the back of an office in Chapter 6: The Fall, Spoiler:there is a secret door hiding a hallway which leads to a dry dock containing a life preserver from The Borealis, a scientific research vessel owned by Aperture Science. The Borealis was first featured in Half Life 2 Episode 2. Getting close to and looking at the life preserver will get you the achievement.
Contributed by Outofmind23
The Turret Opera, the song that was played at the end of the game, was sung entirely by Ellen McLain, who voiced both GlaDOS and the turrets. When recording the song, Ellen was asked to make up some words to put in the song, which she did using her "very bad high school Italian."
Contributed by gamemaster1991
In Chamber 7, there is some sort of robotic singing coming from the Companion Cubes.
Contributed by Hufff
When the Companion Cube is destroyed, you can hear human screaming as it disintegrates. This, and GLaDOS' insistence that the cubes do not talk in the first game, as well as Doug Rattman being able to hear the cube speak, suggest that the cubes contain people.
Contributed by Hufff
In the final Doug Rattmann den in Portal 2, which is accessible through chapter 3 in test 17, if you go near the graffiti art you can hear somebody rambling through the wall, which is believed to be Doug himself.
In test chamber 8, GLaDOS will leave shortly after you start the test to fix the turbines. Before leaving, she will give you sped up instructions. If you slow down what she says, you will hear she is actually quoting a line from Moby Dick.
Contributed by gamemaster1991
Pneumatic Diversity Vent was introduced in a trailer at E3 as a way to move stuff around like turrets. However nowhere in the game can it be found. It was cut because the puzzle aspect was very limiting.
Contributed by Outofmind23
According to Portal 2 writer Erik Wolpaw, Valve originally planned to include a gel that let you walk on walls.

Unfortunately, players would become queasy with its use. Valve tried reducing the frame rate and camera movement, but were eventually forced to drop the feature entirely.
Contributed by IkiFoo
The original idea for the game was to replace Chell with a new protagonist named Mel. Cave Johnson was to replace GLaDOS as the antagonist, and Mel was going to have many different personality spheres follow her (such as the Adventure Sphere, Betty, and one referred to as the "Morgan Freeman Sphere") instead of just Wheatley. Play testers were up for the idea of Mel until they became disappointed when GLaDOS woke up and didn't recognize the player as the character that defeated her in the first game. Mel was then put in the multiplayer before being scrapped entirely and replaced with Atlas and P-Body.
Contributed by gamemaster1991
An unused soundfile for the Announcer exists in Portal 2.
He says: "We will attempt to comply with these now non-existent agencies by playing some more smooth jazz."
No smooth jazz will play after that.

However, in the German version, the music can be heard.
Contributed by SuperFlorian12
In chapter 5, there is a turret that you can rescue for being "different." This turret (named the Oracle Turret by fans and programers) will spout off some several cryptic messages that relate to the later in the game:
• "Get mad. Don't make lemonade." - This is a quote from Cave Johnson.
Spoiler:"Prometheus was punished by the gods for giving the gift of knowledge to man. He was cast into the bowels of the earth and pecked by birds." - This is referencing how Wheatly is given all of GLaDOS's power and turns GLaDOS into a potato, throws her and you into the underground labs, and GLaDOS is pecked at by a bird.
Spoiler:"It won't be enough." - This reveals that the paradox will not work on Wheatly.
Spoiler:"The answer is beneath us." - This refers to the underground labs.
Spoiler:"Her name is Caroline." - Caroline being GLaDOS's real name.
Contributed by gamemaster1991
In one of Ratman's rooms is a radio blaring strange noises. This "noise" is actually a SSTV signal. If you decode it, it will show an image from the end of the game.
Contributed by Chronowarhead
During the "Bring Your Daughter to Work Day" science-fair, you will find a giant potato that is growing to the ceiling. If you look around the potato, you will find that it was made by Chell and she used a "special ingredient from daddy's work." This indicates that Chell was the daughter of an Aperture Science employee.
Contributed by gamemaster1991
During the development of the game, the team stated that there would be no cake jokes, as they were tired of the "The Cake Is A Lie" meme. Eventually, it turns out that they lied about this, as they "couldn't resist putting in just one."
Contributed by gamemaster1991
Because of the failure of the attempted meme of Hoopy the Hoop in Portal, VALVe included the model of Hoopy the Hoop in 3 spots in Portal 2: Near the deactivated GLaDOS in Chapter 1, In Chapter 2 Chamber 2, and behind a desk in a Ratman Den.
Contributed by ORWELL6
In the Spanish version of Portal 2, at the end of chapter 5, when Wheatley takes over GLaDOS' body, Wheatley tries to show his intelligence to Chell by using the translator and in doing so, winds up saying, "It seems that you are using this translation too incorrectly. Please check the manual."
Contributed by Rivalremi
During development, Cave Johnson's "secretary" was a scientist named Greg. But, Valve realized that they would have to hire another voice actor. In order to cut costs and enticed to create an origin for GLaDOS, Greg was cut and Caroline was added. Greg was later used as Cave Johnson's new partner in the Perpetual Testing Initiative DLC.
Contributed by ORWELL6
There are unused audio files in Portal 2 that suggest there was once a scene where Caroline was forcibly transformed into GLaDOS.

'No listen to me! Sir, I do not want this.'
'I do not want this!'
'Mr. Johnson, I don't want this!'
'Cave, I'm begging you, don't! Please!'
*GLaDOS voice* 'I am awake. And I am going to kill you all.'
Contributed by WilbosWorld
Early in development, Portal 2 was planned to feature no portals at all. Valve focused on a new gameplay mechanic called "F-Stop", and though the new mechanic received some positive feedback, every single play tester was disappointed with the absence of portals. Valve then revised the game to include portals.
Contributed by DidYouKnowGaming