Thrombopoietin (TPO), the primary regulator of platelet production, also plays an important role in hematopoietic stem cell (HSC) biology. In previous studies we demonstrated that the self-renewal and expansion of HSCs is 10 to 20 times less robust in tpo^–/– mice than in controls. To explore the molecular basis of this effect, we postulated that Hoxb4 might mediate at least part of the TPO effect on these cells. We first analyzed the effects of TPO on Hoxb4 expression in primitive hematopoietic cell lines; TPO increased expression of the gene 2- to 3-fold in EML and UT-7/TPO cells. We also compared Hoxb4 levels in a candidate HSC population derived from tpo^–/– and control mice; Hoxb4 expression was 2- to 5-fold lower in null HSCs. Of the numerous signal transduction molecules induced by TPO, we found that p38 mitogen-activated protein kinase (MAPK) was responsible for the TPO-induced Hoxb4 elevation. We also demonstrated that upstream stimulating factor 1 (USF-1), a transcription factor previously shown to regulate Hoxb4 expression, is also induced by TPO in a p38-dependent manner. Together, these data provide a molecular pathway by which a growth factor can modulate a transcription factor and thereby help direct a critical developmental process.