The clinical use of embryonic stem cell (ESC)-derived hematopoietic progenitors (ESHPs) requires the generation of ESHPs that produce mature hematopoietic cells and do not induce immune rejection after transplantation. We compared the developmental maturity and immunogenicity of ESHPs generated via two methods: embryoid body (EB) formation and culture of ESCs with the OP9 bone marrow stromal cell line (ESC-OP9). ESHPs derived from EBs displayed an immature hematopoietic phenotype and were devoid of immunogenicity marker expression. In contrast, ESHPs derived via ESC-OP9 displayed a mature phenotype and expressed high levels of some immunostimulatory molecules. ESHPs alone could not stimulate CD4+ T lymphocyte proliferation directly. However, preferential phagocytosis of ESHPs and T cell proliferation were observed in the presence of antigen-presenting cells, consistent with a model of indirect immune recognition of ESHPs. These results suggest that depletion of host CD4+ T lymphocytes or antigen-presenting cells may be necessary for successful ESHP transplantation.