Emerging evidence suggests that all hematopoietic and endothelial cells originate from Flk-1(+) mesoderm in the mouse. However, this concept has not been completely proven, especially for the origin of blood cells. Using either Flk1(+/Cre);Rosa26R-EYFP or Flk1(+/Cre);Rosa26R-LacZ mice, we permanently marked Flk-1(+) cells and their progenies to determine the relationship between hematopoietic tissues and cells that express Flk-1. In embryos, all blood cells within the yolk sac and aorta were of Flk-1(+) origin. In addition, nearly all CD45(+) cells in bone marrow and circulating blood in adults were of Flk-1(+) origin. These results provide clear evidence that all blood cells, primitive and definitive, in mice are derived from Flk-1(+) mesodermal cells.