<b>Background:</b> The de novo generation of patient-specific hematopoietic stem and progenitor cells from induced pluripotent stem cells (iPSCs) has become a promising approach for cell replacement therapies in the future. However, efficient differentiation protocols for producing fully functional human hematopoietic stem cells are still missing. In the mouse model, ectopic expression of the human homeotic selector protein HOXB4 has been shown to enforce the development of hematopoietic stem cells (HSCs) in differentiating pluripotent stem cell cultures. However, the mechanism how HOXB4 mediates the formation of HSCs capable of long-term, multilineage repopulation after transplantation is not well understood yet. <b>Methods:</b> Using a mouse embryonic stem (ES) cell-based differentiation model, we asked whether retrovirally expressed HOXB4 induces the expression of <i>Runx1/AML1</i>, a gene whose expression is absolutely necessary for the formation of definitive, adult HSCs during embryonic development. <b>Results: </b>During ES cell differentiation, basal expression of <i>Runx1</i> was observed in all cultures, irrespective of ectopic HOXB4 expression. However, only in those cultures ectopically expressing HOXB4, substantial amounts of hematopoietic progenitors were generated which exclusively displayed increased <i>Runx1</i> expression. <b>Conclusions:</b> Our results strongly suggest that HOXB4 does not induce basal <i>Runx1</i> expression but, instead, mediates an increase of <i>Runx1</i> expression which appears to be a prerequisite for the formation of hematopoietic stem and progenitor cells.