There's an unused texture file in the game's code referencing the film "300". It can be found in the game's data as "wet_floor_sign.vtf."

In the Normal Test Chamber 17 there is a piece of the level that's out of bounds. It was only meant to be accessed in the Advance Test Chamber 17, which is unlocked after the game is completed. It contains a momentum test with a glass panel that has a hole in the middle. This room can be accessed with cheats by typing 'noclip' into the console. It is also possible to access the room without cheats by using the 'Portal edge Glitch' on PC or it can be accessed on console and pc using a glitch called 'Portal clipping'.

At the end of Portal, Spoiler:when GLaDOS is disconnected and laying in the driveway, a hoop comes out of the air. This was VALVe's attempt at a new meme. Not knowing that "The Cake Is A Lie" would become the resident Portal meme, Hoopy the Hoop was lost into obscurity. The developers consider this the greatest disappointment in Portal.

Portal started out as a student project called "Narbacular Drop." The game was about a princess navigating a dungeon by placing portals in order to escape. The game caught Valve developer Robin Walker's attention, who hired the students to rework the project as Portal.

There are a couple of areas in the game where you can trap yourself in a test chamber with no way to get out. Once the developer figured out that the player could do this, they programed GLaDOS to open an escape route which earned an achievement called "Like A Rat."

GLaDOS had a few different designs before the final one seen in the game. Earlier designs included a floating brain, a sprawling, spidery mechanism, and an upside-down version of Botticelli's painting "The Birth of Venus" made out of robot parts. The final version was a huge mechanical device with a delicate robotic figure, which shows off her raw power and femininity.

There is an unused red portal that can be found in the files of the game.

Throughout the game, a heavily corrupted cake recipe will scroll across various computer monitors. On the second to last level of the game, a GLaDOS memory core will also recite the recipe. Many of the ingredients on the list are real Black Forest Cake ingredients, however there are oddball ingredients including rhubarb, polyester resins and many miscellaneous fish-shaped objects.

The voice of the robot GLaDOS from Portal, Ellen McLain, is the real life wife of John Patrick Lowrie; the voice of the Sniper in Team Fortress 2, another Valve developed game.

In the observation rooms, the keyboards have the letters A, S, D, E, R, H, N, I, and P highlighted in yellow. Unscrambling the letters spells "Adrian Shephard" who is the protagonist of Half-Life: Opposing Force.

A presentation about financial competition between Aperture Science and Black Mesa can be seen in-game Spoiler:while exploring the Aperture offices just before the boss room with GlaDOS, setting the game within the Half-Life universe.