The transcription factor CCAAT/enhancer binding protein alpha, or C/EBPalpha, encoded by the CEBPA gene, is crucial for the differentiation of granulocytes. Conditional expression of C/EBPalpha triggers neutrophilic differentiation, and Cebpa knockout mice exhibit an early block in maturation. Dominant-negative mutations of CEBPA have been found in some patients with acute myeloid leukemia (AML), but not in AML with the t(8;21) translocation which gives rise to the fusion gene RUNX1-CBF2T1 (also known as AML1-ETO) encoding the AML1-ETO fusion protein. RUNX1-CBF2T1 positive-AML blasts had eight-fold lower CEBPA RNA levels and undetectable C/EBPalpha protein levels compared with other subgroups of AML patients. Conditional expression of RUNX1-CBF2T1 in U937 cells downregulated CEBPA mRNA, protein and DNA binding activity. AML1-ETO appears to suppress C/EBPalpha expression indirectly by inhibiting positive autoregulation of the CEBPA promoter. Conditional expression of C/EBPalpha in AML1-ETO-positive Kasumi-1 cells results in neutrophilic differentiation. We suggest that restoring C/EBPalpha expression will have therapeutic implications in RUNX1-CBF2T1-positive leukemias.