Hematopoietic stem cells (HSCs) giv e rise to all cells of the hematopoietic sy stem and are classically defined by their ability to stably engraft and reconstitute the blood sy stem of ablated recipients after transplantation. T his unique function of HSCs is currently exploited in the clinic to treat both patients with hematological diseases, as well as cancer patients who receive high doses of ionizing radiation. The first transplantable HSCs arise from hemogenic endothelium at embry onic day 1 0.5 (E1 0.5) of mouse development and migrate to the fetal liv er (FL), where they undergo a robust expansion followed by a second migration to the fetal bone marrow (FBM) and spleen, starting at E15.5. Our laboratory is currently focused on dissecting the role of these developmental niches in the dynamics of HSC regulation, as this will be critical for defining niche factors essential for optimization of HSCs for therapeutic purposes, including ex vivo expansion/manipulation, as well as enhanced homing and engraftment in transplantation. Current dogma suggests that only a small number of newly specified HSCs are generated during development, yet, identification of these nascent HSCs has been based on functional assays dependent on disruption/manipulation of embryos and readout through transplantation. However, we recently determined that life-long hematopoiesis is initiated by hundreds of independent hematopoietic stem and progenitor cells (HSPCs) during development using an approach that av oids embry o disruption (Ganuza et al. Nature Cell Biology , 201 7). Our recent work revealed that although the FL contains thousands of cy cling and ex panding HSPCs, only sev eral hundreds of these cells establish the adult blood system. Thus, our recent work suggests the existence of a previously unidentified developmental checkpoint downstream of the FL that restricts the ultimate clonal complexity of the adult hem atopoietic sy stem. We hypothesize that the m igration of HSPCs from the FL to FBM represents this checkpoint. To test this, we will determine the absolute number and clonal complexity of bone marrow HSPCs throughout fetal and prenatal development, as well as identify and molecularly define HSPCs capable of colonizing the fetal bone marrow niche. We will perform limi ting dilution analy ses using bone marrow from E17.5 through post-natal day 21, and also analyze the clonal complexity of HSPCs during this dev elopmental window using our nov el Cre rec ombinase ac tivated multi-c olor reporter-based approach. We will also use droplet-based single-cell RNA-Sequencing to interrogate the global molecular profiles of HSPCs in the FL and FBM at pre- and post-migratory timepoints. This work will further illuminate our understanding of the dynamics of hematopoiesis within the early developing bone marrow, an area of research that has been largely unex plored. Identifying nov el fac tors inv olved in the migration of hematopoietic progenitors that are c ritic al for stable, lifelong hematopoiesis will be crucial for optimizing HSPCs for therapeutic purposes, including ex vivo manipulation for expansion and/or enhanced homing and engraftment during transplantation.