The development and differentiation of macrophages in the fetal mouse yolk sac were studied morphologically in four different culture experiments. In the culture of mouse embryos with yolk sac, the development of fetal macrophages was demonstrated to precede that of promonocytes and monocytes in the yolk sac. In vitro differentiation of the fetal macrophages was consistent with the results of our previous in vivo observation indicating that fetal macrophages were differentiated from primitive macrophages, but not from the monocytic cell series. Differentiation of primitive macrophages into fetal macrophages, before the development of promonocytes and monocytes, was reproduced in the culture of cell suspensions from the fetal mouse yolk sacs, with a mouse bone marrow stromal cell clone (ST2) particularly with those at 8 days of gestation. In the soft agar or liquid culture of yolk sac cells with LP3-conditioned medium, monocyte-macrophage colonies were effectively induced, but not fetal macrophage colonies. The results provide evidence for the existence, in yolk sac hematopoiesis, of two distinct macrophage populations: a fetal macrophage population and a monocyte-derived macrophage population. The data indicate an obvious difference in development and differentiation between the two populations and the temporal precedence of fetal macrophages appearing before monocyte-macrophages.