Several lines of evidence suggest that the adult hematopoietic system has multiple developmental origins, but the ontogenic relationship between nascent hematopoietic populations under this scheme is poorly understood. In an alternative theory, the earliest definitive blood precursors arise from a single anatomical location, which constitutes the cellular source for subsequent hematopoietic populations. To deconvolute hematopoietic ontogeny, we designed an embryo-rescue system in which the key hematopoietic factor Runx1 is reactivated in Runx1-null conceptuses at specific developmental stages. Using complementary in vivo and ex vivo approaches, we provide evidence that definitive hematopoiesis and adult-type hematopoietic stem cells originate predominantly in the nascent extraembryonic mesoderm. Our data also suggest that other anatomical sites that have been proposed to be sources of the definitive hematopoietic hierarchy are unlikely to play a substantial role in de novo blood generation.