Donald Duck made his debut in The Wise Little Hen
June 9, 1934
According to Leonard Maltin in his introduction to The Chronological Donald, Donald was created by Walt Disney when he heard Clarence Nash doing his duck voice while reciting Mary had a little lamb. Mickey Mouse had lost some of his edge since becoming a role model for children and Disney wanted a character that could portray some of the more negative character traits he could no longer bestow on Mickey.
Donald Duck first appeared in the cartoon The Wise Little Hen which was part of the Silly Symphonies series of theatrical cartoon shorts.
The Wise Little Hen