Mutations in the CEBPA gene are present in 7%–10% of human patients with acute myeloid leukemia (AML). However, no genetic models exist that demonstrate their etiological relevance. To mimic the most common mutations affecting CEBPA—that is, those leading to loss of the 42 kDa C/EBPα isoform (p42) while retaining the 30kDa isoform (p30)—we modified the mouse Cebpa locus to express only p30. p30 supported the formation of granulocyte-macrophage progenitors. However, p42 was required for control of myeloid progenitor proliferation, and p42-deficient mice developed AML with complete penetrance. p42-deficient leukemia could be transferred by a Mac1⁺c-Kit⁺ population that gave rise only to myeloid cells in recipient mice. Expression profiling of this population against normal Mac1⁺c-Kit⁺ progenitors revealed a signature shared with MLL-AF9-transformed AML.