Significance Acute myeloid leukemia (AML) is a highly heterogeneous form of cancer that results from the uncontrolled proliferation of primitive immune cells. Homeobox A9 (HOXA9) is an evolutionarily conserved transcription factor that is overexpressed in a large percentage of AML cases and is associated with a poor prognosis. Here, we show that CCAAT/enhancer binding protein alpha (C/EBPα), a transcription factor involved in immune cell development that is commonly mutated in AML, is a critical collaborator required for HOXA9-mediated leukemic transformation. We also establish that the cell cycle regulator cyclin-dependent kinase inhibitors Cdkn2a/b are corepressed by the Hoxa9–C/EBPα complex. These findings suggest a novel functional interaction between two leukemic transcription factors, HOXA9 and C/EBPα, that is altered in a large percentage of AML cases.