Mark Hamill reprises his role as The Trickster, which he played in The Flash (1990). The character was even drawn to resemble him as he appeared in Jay and Silent Bob Strike Back (2001).
Flash mentions his uncle coming down for Flash Appreciation Day/Flash Museum opening. In the comics, Wally's uncle was Barry Allen, the second Flash and Wally's mentor during his tenure as Kid Flash. The blonde lab tech with the brush cut hair Wally hands his work to at the start of the show where he works as a CSI - a job Barry Allen actually had in the comics while Wally was a mechanic - may have been drawn to reference him.
In the Flash Museum, the first Flash (Jay Garrick)'s helmet is on display, as well as the costume of Kid Flash (Wally West's previous hero name before donning the title of Flash).
The ring The Flash has collected for the purpose of storing his suit marks the first appearance of a similar ring he used in the same way in the comics.
In some form or another, almost every major villain in the Flash's history appears here, making cameos either at the Rogues' hangout or in depictions at the Flash Museum. The rogues at the bar include Weather Wizard, Turtle-Man, Thinker, Fiddler, Doctor Alchemy, Abra Kadabra, and Pied Piper.