The role of the fetal spleen in hematopoeisis remains largely unknown. In this particular environment, we show that hematopoietic stem cells do not proliferate, but that they lose multipotency and differentiate exclusively into mature macrophages. B lymphocytes in the spleen derive from committed B cell precursors that are likely to have immigrated from the fetal liver. We developed fetal spleen stromal cell lines that are unique in their capacity to expand myeloid precursors, resulting in large numbers of mature macrophages. These lines secrete high levels of anti-inflammatory molecules. By phenotype,fetal splenic macrophages are reminiscent of their adult counterparts found in the red pulp. We postulate that F4/80+ splenic macrophages participate in fetal erythropoiesis, as well as in the formation of the splenic architecture.