The variation of HoxB4 expression levels might be a key regulatory mechanism in the differentiation of human embryonic stem cell (hESC)-derived hematopoietic stem cells (HSCs). In this study, hESCs ectopically expressing high and low levels of HoxB4 were obtained using lentiviral gene transfer. Quantification throughout differentiation revealed a steady increase in transcription levels from our constructs. The effects of the two expression levels of HoxB4 were compared regarding the differentiation potential into HSCs. High levels of HoxB4 expression correlated to an improved yield of cells expressing CD34, CD38, the stem cell leukemia gene, and vascular epithelium-cadherin. However, no improvement in myeloid cell maturation was observed, as determined by colony formation assays. In contrast, hESCs with low HoxB4 levels did not show any elevated hematopoietic development. In addition, we found that the total population of HoxB4-expressing cells, on both levels, decreased in developing embryoid bodies. Notably, a high HoxB4 expression in hESCs also seemed to interfere with the formation of germ layers after xenografting into immunodeficient mice. These data suggest that HoxB4-induced effects on hESC-derived HSCs are concentration-dependent during in vitro development and reduce proliferation of other cell types in vitro and in vivo. The application of the transcription factor HoxB4 during early hematopoiesis from hESCs might provide new means for regenerative medicine, allowing efficient differentiation and engraftment of genetically modified hESC clones. Our study highlights the importance of HoxB4 dosage and points to the need for experimental systems allowing controlled gene expression. Disclosure of potential conflicts of interest is found at the end of this article.