The first definitive/adult-type hematopoietic stem cells (HSCs) in the mouse aorta-gonad-mesonephros region emerge between embryonic days 10.5 and 11.5. The discovery of clusters of hematopoietic cells on the ventral luminal surface of the dorsal aorta in various vertebrate species has led to speculation that the floor of the dorsal aorta may play an essential role for the development of the definitive hematopoietic system. Here, we functionally show affiliation of definitive HSCs with the ventral floor of the dorsal aorta, whereas colony-forming hematopoietic activity is associated with both ventral and dorsal domains. We show that a rare population of PECAM1(high)CD45(+) cells, within which definitive HSCs reside, is predominantly localized to intraaortic clusters. Furthermore, using ex vivo culture analysis, we demonstrate that the ventral domain of the dorsal aorta has an exclusive functional capacity of inducing and expanding definitive HSCs.