The liver maintains hematopoietic stem cells (HSCs) during development. However, it is not clear what cells are the components of the developing liver niche in vivo. Here, we genetically dissected the developing liver niche by systematically determining the cellular source of a key HSC niche factor, stem cell factor (SCF). Most HSCs were closely associated with sinusoidal vasculature. Using Scfgfp knockin mice, we found that Scf was primarily expressed by endothelial and perisinusoidal hepatic stellate cells. Conditional deletion of Scf from hepatocytes, hematopoietic cells, Ng2+ cells, or endothelial cells did not affect HSC number or function. Deletion of Scf from hepatic stellate cells depleted HSCs. Nearly all HSCs were lost when Scf was deleted from both endothelial and hepatic stellate cells. The expression of several niche factors was down-regulated in stellate cells around birth, when HSCs egress the developing liver. Thus, hepatic stellate and endothelial cells create perisinusoidal vascular HSC niche in the developing liver by producing SCF.