HB24 is a diverged homeobox gene known to be expressed in hematopoietic progenitor cells. We show here that the inhibition of HB24 expression in CD34+ bone marrow cells via antisense (AS) oligonucleotides impaired the proliferation of these cells in response to interleukin-3 and granulocyte-macrophage colony-stimulating factor. The treatment of CD34+ cells with HB24 AS oligonucleotides also reduced the levels of c- fos, c-myc, c-myb, cyclin B, and p34cdc2 messenger RNAs compared with cells treated with control oligonucleotides. Conversely, the transient transfection of HB24 into a subpopulation of CD34 cells inhibited their differentiation into mature hematopoietic cell types. In addition, HB24 messenger RNA transcripts were elevated in bone marrow and peripheral blood mononuclear cells isolated from patients with acute myelogenous leukemia compared with normal controls. These data suggest that HB24 is an important transcription factor during hematopoietic progenitor proliferation and that differentiation to specific cell types requires its downregulation. Furthermore, dysregulated expression of HB24 impairs the normal differentiation of hematopoietic progenitors and may contribute to leukemogenesis.