We reported recently that cobalt chloride-simulated hypoxia and mild hypoxia modified the differentiation of human acute myeloid leukemic (AML) cells, probably acting via a hypoxia-inducible factor-1 alpha (HIF-1α)-dependent mechanism. In this study, we investigated the effect of desferrioxamine (DFO), an iron chelator with ‘hypoxia-mimetic’activity, on the differentiation of AML cells. The results showed that DFO at nontoxic concentrations induced the differentiation of AML cell lines NB4 and U937, as assessed by morphological criteria and differentiation-associated antigens. DFO-induced differentiation parallel to the rapid accumulation of HIF-1α protein in these two cell lines. Of importance, the transient transfection of HIF-1α cDNA induced U937 cells to develop the differentiation-related alterations such as growth arrest and increased CD11b expression. Furthermore, the inducible expression of chromosome translocation t (8; 21)-generated leukemogenic AML1-ETO fusion gene attenuated DFO-induced differentiation of U937 cells with the decrease of CCAAT/enhancer-binding protein alpha (C/EBPα), a critical factor for granulocytic differentiation. Using immunoprecipitation and luciferase reporter assay, HIF-1α was also shown to interact physically with and to increase the transcriptional activity of C/EBPα. Taken together, these results provided novel evidence for a role of HIF-1α in AML cell differentiation, and suggested that C/EBPα might be a downstream effector for HIF-1α-mediated differentiation.