During the process of normal hematopoiesis, proliferation is tightly linked to maturation. The molecular mechanisms that lead to production of mature effector cells with a variety of phenotypes and functions from a single multipotent progenitor are only beginning to be elucidated. It is important to determine how these maturation events are regulated at the molecular level, because this will provide significant insights into the process of normal hematopoiesis as well as leukemogenesis. Transcription factors containing the highly conserved homeobox motif show considerable promise as potential regulators of hematopoietic maturation events. In this study, we focused on identification and characterization of homeobox genes of the HOX family that are important in regulating normal human myeloid differentiation induced by the hematopoietic growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF). We have identified three homeobox genes, HOX A5, HOX B6, and HOX B7, which are expressed during early myelopoiesis. Treating bone marrow cells with antisense oligodeoxynucleotides to HOX A5 resulted in inhibition of granulocytic/monocytic hematopoiesis and increased the generation of erythroid progenitors. Also, overexpression of HOX A5 inhibited erythroid differentiation of the K562 cell line. Based on these observations, we propose that HOX A5 functions as an important regulator of hematopoietic lineage determination and maturation.