The Three Little Wolves made their first appearance in the aptly-named short Three Little Wolves. At the beginning of the short, their father is shown educating them on the different food products that can be made out of pigs. Not unlike Huey, Dewey, and Louie, the three wolves are somewhat rebellious, as they slingshot Big Bad when his back is turned. They assist their father in his plan to capture Fiddler and Fifer, where he disguises himself as Little Bo Peep and they dress as her sheep to lure the two gullible pigs to their den. Once in the den, the Three Little Wolves ambush the two pigs, with one wolf chasing Fifer with an axe and the other two Wolves fighting over Fiddler. As Big Bad prepares to cook the pigs, one of the Little Wolves takes the wolf alarm horn that Fifer had brought along and starts playing with it. The pigs tease the little wolf about his horn-blowing until Big Bad takes the horn himself and blows it hard enough to catch Practical's attention. Practical manages to lure Big Bad into his recently-complete Wolf Pacifier machine, which eventually blasts the Wolf into the sky. The Little Wolves see their dad getting launched into the sky and run off to catch him.
The Little Wolves appeared again in The Practical Pig, once again eager to eat Fiddler and Fifer after their father successfully captures them. This time, however, Big Bad Wolf resolves that they will not eat them until after he has also captured Practical. The Little Wolves' sneaky rebelliousness from the previous short is shown off even more here, as they disobey their father's orders and try to eat Fiddler and Fifer early anyway. However, right before they can cook the two pigs as a big pie, they remember that they forgot to add pepper to it. One of them shakes too much pepper over the pigs' noses, causing them to sneeze the pie dough off and onto the Wolves, getting them stuck on the wall and allowing the pigs to escape.
The two animated shorts featuring the Three Little Wolves were later adapted, along with the original Three Little Pigs short, on a vinyl record album in 1967. Their portrayal on the album differs quite a bit from the shorts, possibly as a result of the creation of Li'l Wolf in the comic books. Unlike the cartoons, the album refers to the Three Little Wolves (or at least two of them) as the Big Bad Wolf's nephews instead of his sons. Also, one of them is depicted as being just like (or is actually supposed to be) Li'l Wolf, to the point that he helps Fiddler and Fifer escape at the end of The Practical Pig segment.
The Three Little Wolves' (so far) only new appearance in animation since the Silly Symphonies series ended was in the House of Mouse episode "Pete's House of Villains". When Pete takes control of the club and puts his fellow Disney Villains in charge of all the crew positions, the Three Little Wolves had a cameo, replacing Huey, Dewey, and Louie as the house band. Interestingly, that episode also featured a new cartoon short starring Li'l Wolf; therefore, it is unclear if, in the context of House of Mouse, the Three Little Wolves are still considered Big Bad Wolf's sons or if they are his nephews as stated in the above-mentioned album.