Inability to generate self-renewing hematopoietic stem cells from pluripotent stem cells (PSC) or via reprogramming has proven to be a major bottleneck that inhibits the use of in vitro engineered hematopoietic cells for therapeutic purposes. We hypothesize that defective induction of the correct transcriptional networks governing HSC self-renewal during the critical stages of hematopoietic development prevents the emergence of fully functional HSC in an in vitro setting. Guided by the transcriptional profile of highly purified human fetal liver HSC, we aim to identify key transcriptional regulators that govern self-renewal in developing human HSC and use this knowledge to develop new strategies to improve the function of in vitro derived hematopoietic cells. To identify the best candidates for self-renewal regulators, we took advantage of a novel human HSC surface marker, GPI80, to purify a population that is highly enriched for the true self-renewing HSC during human development. This strategy facilitated the identification of a set of transcriptional regulators that are unique to the self-renewing GPI80 HSC as compared to their downstream progeny, and are also suppressed in PSC-HPC. Utilizing our newly defined HSC culture system, which allows the study of human multilineage HSPC (hematopoietic stem/progenitor cell) hierarchy in vitro, we will first perform lentiviral knockdown of these candidate HSC regulators in primary human fetal liver GPI80+ HSC to test their importance for protecting HSC self-renewal and multipotency. In parallel, we will also investigate the degree to which the induction of these candidate self-renewal regulators extends HSC activity in cultured HSC, and/or even converts the transcriptional program in non-self-renewing GPI80-HPC, bringing them functionally and molecularly closer to fetal liver HSC. Finally, we will define the hierarchy of transcriptional networks associated with HSC self-renewal by analyzing the genomic binding sites and downstream gene expression networks for the individual transcriptional regulators that are involved in maintaining HSC self-renewal. Identification of the key upstream regulators that can confer self-renewal in developing HSC will both increase our knowledge of the fundamental regulatory mechanisms governing human HSC as well as pave the way for developing novel approaches for the in vitro generation of HSC for therapeutic use.