Hematopoietic stem cell (HSC) generation in the aorta-gonad-mesonephros region requires HSC specification signals from the surrounding microenvironment. In zebrafish, PDGF-B/PDGFRβ signaling controls hematopoietic stem/progenitor cell (HSPC) generation and is required in the HSC specification niche. Little is known about murine HSPC specification in vivo and whether PDGF-B/PDGFRβ is involved. Here, we show that PDGFRβ is expressed in distinct perivascular stromal cell layers surrounding the mid-gestation dorsal aorta, and its deletion impairs hematopoiesis. We demonstrate that PDGFRβ⁺ cells play a dual role in murine hematopoiesis. They act in the aortic niche to support HSPCs, and in addition, PDGFRβ⁺ embryonic precursors give rise to a subset of HSPCs that persist into adulthood. These findings provide crucial information for the controlled production of HSPCs in vitro.