HOXB4, a member of the Homeobox transcription factor family, promotes expansion of hematopoietic stem cells and hematopoietic progenitor cells in vivo and ex vivo when overexpressed. However, the molecular mechanisms underlying this effect are not well understood. To identify direct target genes of HOXB4 in primary murine hematopoietic progenitor cells, we induced HOXB4 function in lineage-negative murine bone marrow cells, using a tamoxifen-inducible HOXB4-ER(T2) fusion protein. Using expression microarrays, 77 probe sets were identified with differentially changed expression in early response to HOXB4 induction. Among them, we show that Hemogen (Hemgn), encoding a hematopoietic-specific nuclear protein of unknown function, is a direct transcriptional target of HOXB4. We show that HOXB4 binds to the promoter region of Hemgn both ex vivo and in vivo. When we overexpressed Hemgn in bone marrow cells, we observed that Hemgn promoted cellular expansion in liquid cultures and increased self-renewal of myeloid colony-forming units in culture, partially recapitulating the effect of HOXB4 overexpression. Furthermore, down-regulation of Hemgn using an shRNA strategy proved that Hemgn contributes to HOXB4-mediated expansion in our myeloid progenitor assays. Our results identify a functionally relevant, direct transcriptional target of HOXB4 and identify other target genes that may also participate in the HOXB4 genetic network.