The study investigates the origin of hematopoietic stem cells (HSCs) during zebrafish development. Using fluorescent reporter transgenes, confocal timelapse microscopy, and flow cytometry, the researchers directly image and isolate the stepwise transition from aortic hemogenic endothelium to nascent HSCs. They demonstrate that HSCs generated from this lineage are the founders of the adult hematopoietic system. The findings support the hypothesis that HSCs arise directly from hemogenic endothelium lining the ventral wall of the dorsal aorta, complementing previous studies in other vertebrates and suggesting conserved mechanisms of HSC generation across evolution. The results have implications for instructing HSC formation *in vitro* from pluripotent precursors, which is crucial for cell replacement therapies.The study investigates the origin of hematopoietic stem cells (HSCs) during zebrafish development. Using fluorescent reporter transgenes, confocal timelapse microscopy, and flow cytometry, the researchers directly image and isolate the stepwise transition from aortic hemogenic endothelium to nascent HSCs. They demonstrate that HSCs generated from this lineage are the founders of the adult hematopoietic system. The findings support the hypothesis that HSCs arise directly from hemogenic endothelium lining the ventral wall of the dorsal aorta, complementing previous studies in other vertebrates and suggesting conserved mechanisms of HSC generation across evolution. The results have implications for instructing HSC formation *in vitro* from pluripotent precursors, which is crucial for cell replacement therapies.