In the basement of the Helicarrier, a character will ask the player to help them clear out some snakes, saying,



S.H.I.E.L.D. Director Nick Fury's appearance in the Marvel Ultimate line of comic books was based on Samuel L. Jackson, and he has played the role in a number of Marvel movies. Samuel L. Jackson also played the part of Neville Flynn, an FBI agent in the film "Snakes on a Plane", in which he exclaims, "That is it! I have had it with these mother-fucking snakes, on this mother-fucking plane!"
The opening line is also likely a reference to Indiana Jones, with the same line appearing in the 1981 movie, Raiders of the Lost Ark.

While playing as Spider-Man, leading up to the boss battle against Venom, Spider-Man will exclaim that enemies are "coming out of the walls!" This is likely a reference to the famous line from the 1986 film "Aliens".

Shortly after his introduction, the Human Torch comments that a certain bridge looks like a rainbow road, and it makes him want to race. This is likely a reference to the Rainbow Road seen throughout the Mario series, primarily as a racing track in the Mario Kart games.

The achievement "Don't I Know You?", which is obtained by controlling both Captain America and Human Torch in free play, is a joke about how Captain America and Human Torch were both portrayed by the same actor (Chris Evans) in Captain America: The First Avenger and the Fantastic Four films, respectively.

At the begining of Level 7, Thor opens a portal to the Bifrost, and the characters are sent flying through a wormhole which greatly resembles those The Doctor travels through in the fourth season of Dr. Who. Further reinforcing the reference are several blue phone booths also travelling through the wormhole, which resemble The Doctor's TARDIS.

In the Deadpool mission "House Party Protocol" when you Spoiler:build the DJ booth Nick Fury says "And you will know I am the DJ when I lay my beats down on you.", this is a reference to a line in Pulp Fiction where Jules Winnfield quotes Ezekiel 25:17; "You will know my name is the lord when I lay my vengeance upon thee."
The actor Samuel L. Jackson played both Jules Winnfield in Pulp Fiction and Nick Fury in the Marvel movies.

In Level 9, "Doctor in the House", Mr. Fantastic uses himself as a parachute. He then says "I saw this in a movie once."

This is a reference to the Pixar film The Incredibles, in which Mrs. Incredible uses her powers in a similar fashion.

If the player has their character stare at the face of the Statue of Liberty, then after a while she will move her head slightly and wink at the player.

Using the Human Torch's flame powers near Venom will have the latter cower in fear in reference to the symbiote's fear of fire in the comics.

In an homage to a classic move consistently done by the duo in numerous media, if the Player attacks Wolverine with Colossus, then Colossus will pick up and throw Wolverine instead of doing his usual attack.

In various locations within the New York hub world, the player can trigger an event that allows them to fight a Mutant-hunting Sentinel from the X-Men line of comics. Defeating the Sentinel unlocks a miniature verison as a playable character. Much like the comics, the Sentinels only target Mutant characters (Wolverine, Magneto, Storm, etc.) though will ignore non-Mutant characters unless provoked.

The achievement/trophy "Menace of Magneto" can be unlocked by piloting the Magneto Mobile as Magneto near the Baxter Building. This is a reference to "The Menace of Magneto", an episode of the 1978 animated series "The New Fantastic Four" where the titular vehicle originated from.