The cellular microenvironments in which dendritic cells (DCs) develop are not known. DCs are commonly expanded from CD34+ bone marrow precursors or blood monocytes using a cocktail of growth factors including GM-CSF. However, cytokine-supported cultures are not suitable for studying the intermediate stages of DC development, since progenitors are quickly driven to become mature DCs that undergo limited proliferation and survive for only a short period of time. This lab has developed a long-term culture (LTC) system from spleen which readily generates a high yield of DCs. Hematopoietic cells develop under more normal physiological conditions than in cultures supplemented with cytokines. A spleen stromal cell monolayer supports stem cell maintenance, renewal, and the specific differentiation of only DCs and no other hematopoietic cells. Cultures maintain continuous production of a small population of small-sized progenitors and a large population of fully developed DCs. Cell–cell interaction between stromal cells and progenitor cells is critical for DC differentiation. The progenitors maintained in LTC appear to be quite distinct from bone marrow–derived DC progenitors that respond to GM-CSF. The majority of cells produced in LTC are large-sized cells with a phenotype reflecting myeloid-like DC precursors or immature DCs. These cells are highly endocytotic and weakly immunostimulatory for T cells. This model system predicts in situ production of DCs in spleen from endogenous progenitors, as well as a central role for spleen in DC hematopoiesis.