PROJECT SUMMARY/ABSTRACT Our long-term research goals are to understand the mechanisms that regulate stem cell fate decisions. In this first renewal application of this award, we propose to pursue the regulation of self-renewal and lineage potential of the developmentally restricted hematopoietic stem cells (drHSCs) that we discovered in the current award period. Although this population fulfills the most stringent criteria for functional HSC, their life-span during normal development is restricted to a limited developmental window. A functional HSC that does not persist into adulthood had never been observed before and therefore defines a novel wave of definitive hematopoiesis with a distinct endpoint. Here, we focus on understanding the contradictory regulation of drHSC self-renewal and multipotency: upon transplantation, drHSC self-renewal is induced, whereas their intrinsic lineage bias is preserved. Amazingly, the latter – lymphoid bias and exceptional B1a reconstitution potential – is maintained over many months even upon the repeated stress of serial transplantation. In contrast, a single, short-term exposure to stress induces the ability of drHSCs to persist long-term. We propose to pursue the epigenetic mechanisms governing this paradox. We will perform comprehensive molecular and cellular comparisons of drHSCs, co-existing fetal liver HSCs, and adult HSCs, and pursue rigorous functional analysis in competitive reconstitution assays. Importantly, we will couple single-cell transcriptional profiles with functional HSC capacity in efficient yet rigorous in vivo assays. Using CRISPRi/a-mediated transcriptional manipulation, we will directly test the requirements for reprogramming HSC self-renewal and lineage potential in vivo. Our transgenic models are uniquely suited for understanding how the core properties of HSCs – self-renewal and multilineage potential - are established during development and maintained for life and we are excited to put these powerful tools to work to pioneer developmental hematopoietic fate decisions.