PROJECT SUMMARY Hematopoietic stem and progenitor cells (HSPCs) are characterized by their self-renewal and multipotent differentiation capacities. As such, they give rise to all mature blood cell types (e.g., myeloid, lymphoid, and erythroid) to maintain life-long hematopoiesis. Their regenerative capacity makes HSPCs valuable for cell replacement therapies in patients with hematological diseases, including those that are secondary to chemotherapy and radiotherapy. Understanding HSPC properties of self-renewal and multipotency allows for the development of methods to improve and maximize their therapeutic potential. By studying the embryonic origins of HSPC self-renewal and differentiation capacities, we aim to advance what is known about these defining characteristics of stem cells. In addition to HSPCs, other multi-lineage progenitors are produced during embryogenesis. These are limited in their self-renewal and differentiation output and are mostly regarded as transient in nature. These progenitor populations share many features of stem cells, confounding studies of HSPC properties within their native embryonic environments. Several studies in murine and zebrafish models suggest that these embryonic progenitors, and not HSPCs, are the dominant population generating mature blood cells in the embryo. If HSPCs are not necessary to sustain the embryo, what is their function during development? To answer this question, we propose to use zebrafish to determine when and where during development HSPCs self-renew and contribute to mature blood cell output in myeloid, lymphoid, and erythroid lineages. We will use novel regeneration and transplantation assays (Aim 1) to study self- renewal and lineage-tracing experiments (Aim 2) to investigate HSPC differentiation. Understanding how these properties are established and maintained is critical to harnessing stem cells for regenerative medicine.