- Iron Man (Tony Stark) (Cameo)
- Uncle Ben Parker (Cameo)
- Aunt May Parker (Cameo)
- Taskmaster (Tony Masters)
- Taskmaster Acolytes
- Hydra (Vision)
- Doctor Doom (Victor von Doom) (Mentioned)
- Daily Bugle
- J. Jonah Jameson (Appears on a computer screen, TV, or hologram only)
- Hailey Wilson (First appearance) (Only in flashback)
- Juggernaut (Cain Marko) (Photo)
- Hawkeye (Clint Barton) (Photo)
- Wolverine (Logan) (Mentioned)
- Spider-Man's Web-Shooters
- Deadpool's Suit
- Deadpool's Katanas
- Nova Helmet
- White Tiger Amulet
- Iron Man Armor (Only in flashback)
- S.H.I.E.L.D. Jet Pack
While web-slinging through New York in the rain Spider-Man discusses his life, but gets splashed with water by a speeding taxi cab. He interrupted by another person who is also able to break the "Fourth Wall": Deadpool!. He fights a group of ninjas in an alley continually making his own sound effects, but easily defeating his opponents, taking one of them out with a wedgie, he explained that he was on a mission where he is looking for the secret headquarters of Taskmaster. As focus returned back to Spider-Man he wondered where everyone went at that moment J. Jonah "Loudmouth" Jameson then screams that "Spider-Man is a menace" and he land in a nearby garbage truck.
On the Tricarrier the team battle robots in a training session lead by Agent Coulson as Spider-Man entered smelling of garbage. Deadpool in the briefing room is told the team of the time where he defeated Doctor Doom single-handedly. Spider-Man interrupted him and they explain that Deadpool was in the program before he was recruited. Deadpool tells Spider-Man that Nick Fury considers him a son and he can prove it he has papers. Spider-Man is insulted that Deadpool thinks he is just a copy cat ripping off his costume. Deadpool then goes into the Tricarrier's computer to look for Agent McGuffin who has stolen the encrypted information about the secret identities of every superhero and S.H.I.E.L.D. agent. He finds the location of the Taskmaster's training camp. Spider-Man trails Deadpool to help him on a mission (and avoid a training session). While riding in "Deadpool's jet", (which is revealed to be Tony Stark's stolen jet, as he left him and I.O.U. message on the runway). While in the jet Deadpool recounts his origins, of being a normal baby for only a second before he was attacked by ninjas, but due to his mental state it's impossible to believe a word he says.
Spider-Man and Deadpool are attacked by Taskmaster' Acolytes an elite force personally trained by him. They used stolen S.H.I.E.L.D. jet packs and attack the craft battling the Acolytes while falling. After dodging the missile, Deadpool reveals he is wearing a parachute as Spider-Man crashes into a tree. Deadpool and Spider-Man enter a nearby compound where Spider-Man works to keep Deadpool from killing Taskmaster students. Taskmaster tries to attack Deadpool to no avail (due to Deadpool's unpredictable moves). Spider-Man manages to web up Taskmaster as it turns out that Taskmaster actually stole the S.H.I.E.L.D. list from Deadpool who planned to sell it for money and made up the "Agent McGuffin". Spider-Man then fights with Deadpool while evading the compound's death traps, Deadpool reveals what is thought to be his true origin but it is impossible to confirm, he was a street kid taken in by Fury and at first the program worked, then he realised he was being manipulated and controlled and decided to leave. Spider-Man decides that Deadpool is not a hero but a mercenary who would do anything for money. Spider-Man then remembers that if it wasn't for Aunt May or Uncle Ben he may have turned out the same. Spider-Man manages to defeat Deadpool in a fantasy fight, involving giant chickens and cows on a farm and a fight dressed as pirates, ending the fight with a "Massive Morality K.O."(a boxing Glove emerging from a book Spider-Man tosses at Deadpool with a spirit voice saying: "With Great Power always Comes with Great Responsibility", a rule Spidey will never forget). Deadpool then gets away using a stolen jet pack, though Spider-Man is able to recover the file.
- In the WatchMojo.com list Top 10 4th Wall Breaks in TV, Deadpool's appearance in this episode was ranked as number one.
- Agent McGuffin is named for a writer's device for motivating the major characters.