The transcription factor C/EBPα is required for granulopoiesis and frequently disrupted in human acute myeloid leukemia (AML). Here, we show disruption of C/EBPα blocks the transition from the common myeloid to the granulocyte/monocyte progenitor but is not required beyond this stage for terminal granulocyte maturation. C/EBPα-deficient hematopoietic stem cells (HSCs) have increased expression of Bmi-1 and enhanced competitive repopulating activity. Bone marrow in adult C/EBPα-deficient mice was filled with myeloblasts, similar to human AML, supporting the notion that disruption of C/EBPα cooperates with other events in the development of leukemia. Therefore, C/EBPα is not only essential for granulocyte development but, in addition, is a regulator of hematopoietic stem cell activity.