SUMMARY Pluripotent stem cells (PSC) are an ideal source for deriving HLA-matched or patient specific hematopoietic stem cells (HSC) for the treatment of blood disorders. However, all the efforts in producing self-renewing hematopoietic stem cells (HSC) from PSC have failed due to our limited understanding of the mechanisms that govern “stemness” in developing human HSC. Although the transcription factors that drive blood specification are relatively well understood, we lack knowledge of the programs that define HSC self-renewal, and why these programs fail in PSC derived hematopoietic cells. Thus, we aim to create a single cell transcriptome map of human HSC ontogeny in vivo and ESC derived cells generated in vitro. The ability to compare human HSC from the critical stages of ontogeny when HSCs are specified (AGM, and possibly the placenta and yolk sac) and expand and mature into fully functional HSC in the conceptus (fetal liver and fetal bone marrow) will allow us to identify pathways that are critical for HSC self-renewal, and that distinguish human HSCs developing in vivo from those that develop in vitro. This analysis will pinpoint key defects that underlie the poor function of ESC derived hematopoietic cells, and offer new solutions for overcoming these molecular barriers. As there is a lack of HSC surface markers that would reliably predict human HSC function, to help monitor the differentiation of properly specified HSCs, we created hESC reporter lines for critical HSC regulatory factors HOXA5, MLLT5 and HLF whose expression is highly enriched in self-renewing human HSCs. We will then compare the in vitro derived candidate HSC to the single cell hemato-vascular lineage map of human hematopoietic tissues as HSCs emerge and expand in vivo. This SHINE-II RO1 is designed to address critical hurdle steps in in vitro HSC generation. These studies can be expanded to mechanistic studies to understand human HSC regulation, and ultimately to translational studies to improve HSC transplantation.