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Previously, we reported generation of erythropoiesis from acetyl low-density lipoprotein (Ac-LDL)- incorporating endothelial cells (ECs) in the mouse embryo. However, it is still unclear whether the other types of definitive hematopoietic cells (HCs) can be generated from these cells. In this study, ECs at 10 days post coitum (dpc) were tagged with Ac-LDL-DiI and were shown to release DiI⁺ HCs into the circulation after 12 h of whole embryo culture. The hematopoietic clusters in the main arteries were also stained with DiI. The circulating DiI⁺ HCs expressed c-Kit and half of these cells displayed blastic morphology. In vitro culture and in vivo reconstitution experiments demonstrated that the circulating DiI⁺ HCs contained definitive multipotent progenitors, including hematopoietic stem cells (HSCs). Furthermore, the sorted DiI⁺ HCs were able to colonize the fetal liver (FL) when introduced back into the blood stream of a secondary recipient embryo. These results suggest that Ac-LDL incorporating ECs can produce definitive HSCs and HCs colonizing FL in the mouse embryo.