Enforced expression of Hoxb4 dramatically increases the regeneration of murine hematopoietic stem cells (HSCs) after transplantation and enhances the repopulation ability of human severe combined immunodeficiency (SCID) repopulating cells. Therefore, we asked what physiologic role Hoxb4 has in hematopoiesis. A novel mouse model lacking the entire Hoxb4 gene exhibits significantly reduced cellularity in spleen and bone marrow (BM) and a subtle reduction in red blood cell counts and hemoglobin values. A mild reduction was observed in the numbers of primitive progenitors and stem cells in adult BM and fetal liver, whereas lineage distribution was normal. Although the cell cycle kinetics of primitive progenitors was normal during endogenous hematopoiesis, defects in proliferative responses of BM Lin(-) Sca1(+) c-kit(+) stem and progenitor cells were observed in culture and in vivo after the transplantation of BM and fetal liver HSCs. Quantitative analysis of mRNA from fetal liver revealed that a deficiency of Hoxb4 alone changed the expression levels of several other Hox genes and of genes involved in cell cycle regulation. In summary, the deficiency of Hoxb4 leads to hypocellularity in hematopoietic organs and impaired proliferative capacity. However, Hoxb4 is not required for the generation of HSCs or the maintenance of steady state hematopoiesis.