Abstract Abstract SCI-44 Hematopoietic stem cells (HSCs) develop from endothelium in the midgestation mouse conceptus, starting at 10.5 days post coitus, through the formation of intra-aortic clusters. Studies in mouse, zebrafish, chick, and frog embryos established that Runx1 (AML1) is the earliest specific marker of the endothelium from which HSCs and definitive hematopoietic progenitors form. Conditional deletion of Runx1 in vascular endothelial cadherin (VEC) positive cells blocks the emergence of intra-aortic hematopoietic clusters, definitive hematopoietic progenitors (HPs), and HSCs, demonstrating that Runx1 is required in endothelial cells for HP and HSC formation. On the other hand, Runx1 deletion in fetal liver HPs and HSCs with Vav-Cre, or deletion in adult HSCs with Mx1-Cre, does not eliminate HP and HSC function, although it does affect homeostasis. These data (and others) demonstrated that the absolute requirement for Runx1 in HP and HSC formation is transient, and over by the time HPs and HSCs colonize the fetal liver. We are further defining the developmental windows during which Runx1, and the gene encoding its non-DNA binding partner CBFβ, are required for HP and HSC specification and commitment. We have taken two complementary approaches. One approach is to delete Runx1 at specific times and assess the effect on HPs. The second approach is to restore CBFβ function at specific times, or in specific cells in otherwise CBFβ deficient embryos and determine whether that rescues HP or HSC formation. From these experiments we can draw several conclusions: 1) Runx1 is absolutely required before the 40 somite pair stage in the AGM region, as deletion before then severely impairs HP formation or activity; 2) CBFβ activity is required before 9.5 dpc for HP and HSC formation; 3) CBFβ activity in endothelium alone is sufficient for HP formation, but insufficient for HSC formation. Together the data indicate that Runx1-CBFβ activity is required over a period of several days during embryogenesis to specific and commit HSCs. Disclosures: No relevant conflicts of interest to declare.