The site of origin of lymphohematopoietic stem cells (HSC) that initiate definitive blood cell production in the murine fetal liver is controversial. Contrary to reports that the preliver yolk sac does not contain definitive HSC, we observed that CD34+ day 9 yolk sac cells repopulated multiple blood cell lineages in newborn hosts for at least 1 year. Furthermore, 100 CD34+c-Kit+ day 9 yolk sac or para-aortic splanchnopleura (P-Sp) cells, known to give rise to embryonic HSC, similarly repopulated hematopoiesis in recipient hosts. Surprisingly, 37-fold more CD34+c-Kit+ cells reside in the day 9 yolk sac than in the P-Sp. In sum, definitive HSC are coexistent, but not equal in number, in the murine yolk sac and P-Sp prior to fetal liver colonization.