Summary The goal of this proposal is to understand how hematopoietic stem cells (HSCs) form in the embryo. All HSCs in the adult bone marrow are descendants of pre-hematopoietic stem cells (pre- HSCs) that differentiate from hemogenic endothelial (HE) cells in the major caudal arteries of the embryo. However, only a subset of hematopoietic cells that differentiate from HE cells in the arteries are pre-HSCs, while many are committed lympho-myeloid biased progenitors. We generated a comprehensive single cell dataset that captures the entire developmental trajectory from arterial endothelial cells to lympho-myeloid biased progenitors and pre-HSCs. This dataset is a powerful tool for generating and testing novel concepts in HSC ontogeny. One discovery from this analysis is an endothelial cell precursor of HE cells that we named pre-HE. Pre-HE cells are a transitional population from which a limited number of cells will be specified as HE. We discovered that the efficiency at which pre-HE cells differentiate into HE cells is determined by the levels of RUNX1, a transcription factor that is expressed in HE cells and is essential for HE cell specification. We also identified a candidate enhancer in the Runx1 gene that first becomes accessible in pre-HE cells. One goal of this proposal is to determine if the Runx1 pre-HE enhancer is required for the differentiation of pre-HE cells into HE cells, and which transcription factors and upstream signaling pathways regulate the activity of this enhancer. A second important finding is that we defined the molecular differences between lympho-myeloid biased progenitors and pre- HSCs, and identified genes specifically expressed in each of these populations. One of these genes, Mecom, which is known to regulate adult HSC proliferation and function, is more highly expressed in pre-HSCs than in lympho-myeloid biased progenitors. We will determine if the expression of Mecom in endothelial cells regulates the number of pre-HSCs that are generated during hematopoietic ontogeny. Understanding how arterial HE cells are specified and specialized will help guide ongoing efforts to generate HSCs from other cell sources.