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In the original short, Steamboat Willie is first seen being steered by Mickey, playing about in Pete's cabin as if he were the captain. Pete catches him in the act and orders him to get back down to the deck. The boat is brought to Podunk Landing where Mickey has to load a cow onto the deck. When Mickey then sees Minnie Mouse wanting to hitch a ride onto Steamboat Willie, he uses the ship's crane to bring her on deck, but the goat on deck eats Minnie's fiddle and sheet music. Mickey and Minnie then spend the afternoon playing "Turkey in the Straw" with all the animals on the boat, but Pete eventually puts an end to this and makes Mickey go peel potatoes as punishment.
At the end of Aladdin and the King of Thieves, Genie exits the mouth of the giant turtle in a Steamboat Willie-like boat, mimicking Mickey in the original short's opening scene.
In the Mickey Mouse Works short "How to Be a Waiter", Steamboat Willie is seen during Goofy's attempt at filming a silent movie. Goofy, mimicking the original's opening scene, steers an identical steamboat and ends up accidentally crashing into Willie.
Steamboat Willie is the subject of a comic story titled "Mickey Mouse and the River of Time", in which the sunken ruins of the ship turn up in a river and Mickey and Pete work to salvage the craft.
At the end of the Disney's Hollywood Studios version of Fantasmic!, Mickey steers Steamboat Willie across the river in front of the mountain, with various Disney characters all on board the ship, dancing with golden streamers. (It should be noted that the original Disneyland version of the show instead uses the Mark Twain Riverboat for this scene.)
As Steamboat Willie is the subject of the first level in Mickey Mania: The Timeless Adventures of Mickey Mouse, the very first part of the level is set on the steamboat. When Mickey comes face-to-face with his younger self from the short, Steamboat Willie Mickey bolts out of the captain's cabin. When Mickey destroys the crane machine at the end of the level, the player is informed that Mickey has saved Steamboat Willie, despite the boat being nowhere near the battle.
In Kingdom Hearts II, Steamboat Willie prominently appears in the Timeless River level. The present-day Pete steals it in order to transport the Cornerstone, but Sora and his crew manage to retake the steamboat and the stone. Later, after helping defeat his future self, the Captain Pete of the past rewards Sora and the others with a ride on his steamboat, because Mickey never showed up for work today.
In Epic Mickey, Mickey traverses across Steamboat Willie in one of the game's 2D transitional stages between worlds.
In the Phineas and Ferb: Across the 2nd Dimension video game, part of the Old-Timey Dimension has the characters visiting a version of Steamboat Willie piloted by that dimension's counterpart of Heinz Doofenshmirtz.