This study demonstrates that hematopoietic stem cells (HSCs) arise directly from hemogenic endothelium in the ventral wall of the dorsal aorta during zebrafish embryonic development. Using fluorescent reporter transgenes, confocal timelapse microscopy, and flow cytometry, the researchers identified and isolated the stepwise intermediates as hemogenic endothelium transitions to nascent HSCs. They also used a permanent lineage tracing strategy to show that HSCs generated from hemogenic endothelium are the lineal founders of the adult hematopoietic system. The study supports the hypothesis that HSCs originate from endothelium or shared endothelial precursors. The findings are consistent with previous studies in avian, amphibian, and mammalian embryos, suggesting that the cellular mechanisms of HSC generation are highly conserved across vertebrate evolution. The study also shows that HSC development requires transition through a hemogenic endothelial intermediate, which should aid efforts to instruct HSC formation in vitro from pluripotent precursors, a necessity for therapies designed to replace the adult blood cell lineages. The study used a combination of genetic and molecular techniques to analyze the development of HSCs, including the use of transgenic zebrafish models and flow cytometry to analyze the expression of hematopoietic and vascular genes. The results indicate that HSCs arise from endothelium and that the transition from endothelium to HSC is regulated by the Runx1 transcription factor. The study also shows that HSCs migrate ventrally towards the caudal vein, consistent with previous observations in zebrafish. The findings provide important insights into the development of HSCs and have implications for regenerative medicine and blood cell therapy.This study demonstrates that hematopoietic stem cells (HSCs) arise directly from hemogenic endothelium in the ventral wall of the dorsal aorta during zebrafish embryonic development. Using fluorescent reporter transgenes, confocal timelapse microscopy, and flow cytometry, the researchers identified and isolated the stepwise intermediates as hemogenic endothelium transitions to nascent HSCs. They also used a permanent lineage tracing strategy to show that HSCs generated from hemogenic endothelium are the lineal founders of the adult hematopoietic system. The study supports the hypothesis that HSCs originate from endothelium or shared endothelial precursors. The findings are consistent with previous studies in avian, amphibian, and mammalian embryos, suggesting that the cellular mechanisms of HSC generation are highly conserved across vertebrate evolution. The study also shows that HSC development requires transition through a hemogenic endothelial intermediate, which should aid efforts to instruct HSC formation in vitro from pluripotent precursors, a necessity for therapies designed to replace the adult blood cell lineages. The study used a combination of genetic and molecular techniques to analyze the development of HSCs, including the use of transgenic zebrafish models and flow cytometry to analyze the expression of hematopoietic and vascular genes. The results indicate that HSCs arise from endothelium and that the transition from endothelium to HSC is regulated by the Runx1 transcription factor. The study also shows that HSCs migrate ventrally towards the caudal vein, consistent with previous observations in zebrafish. The findings provide important insights into the development of HSCs and have implications for regenerative medicine and blood cell therapy.