Iron Man to War Machine: Stop talking like you're on Reading Rainbow!
In "Too Many Wolverines", Egghead tries to stop Reptil with a can of Dinosaur Spray, only for it spit out whipped cream. This is a nod to Wayne Knight's role as Dennis Nedry in Jurassic Park, where he tried to smuggle dinosaur embryos in a can of shaving cream.
Executive Meddling: Despite Disney purchasing Marvel around the time of the show's premiere, Cartoon Network was forced to continue airing the series due to a contractual obligation to Hasbro, who produced the toyline.
Exiled from Continuity; Spider-Man and related characters weren't allowed to appear on the show because Sony still owned the full media rights to Spider-Man when the show was in development. Because Sony didn't relinquish Spidey's animated television rights to Disney yet, the wall-crawler didn't even get to make so much as a cameo in the second season. The closest the series ever gets to acknowledging Spider-Man is having the Mayor of Superhero City mention a hero who gained super powers from being bitten by a radioactive bug in "Election of Evil".
Screwed by the Network: When Season 2 started on Cartoon Network, it only aired on Saturday mornings at 6:30 AM. Disney's purchase of Marvel was likely a factor in this.
Talking to Himself: There are some characters who share voice actors and interact with one another.
Tara Strong voices both H.E.R.B.I.E. and Scarlet Witch.
Tom Kenny voices Iron Man, M.O.D.O.K., and Captain America.
What Could Have Been: Spider-Man was slated to appear in the series during conception, possibly in Falcon's spot, but Sony refused due to their ownership of the TV rights to the character at the time. Even after Sony returned the TV rights to Marvel following Disney's acquisition of said company, he still wasn't able to appear at all in the series. The closest we got was in the Ultimate Spider-Man episode "Flight of the Iron Spider", which shows a SHS-Spidey in one of Spidey's own Imagine Spots as Tony Stark is talking about an invention that can send people into different dimensions which also serves as a Chekhov's Gun as that's how he gets rid of the Living Laser - sending him exactly into SHSS to be beaten up by Thor.
This is somewhat rectified by him (and some other Spider-Heroes) appearing in the online game, which for all intents and purposes is the same universe as the cartoon.