A novel CD11c(lo)CD11b(hi)MHC-II⁻CD8α⁻ dendritic-like cell (L-DC) develops in cocultures of bone marrow over splenic stroma. L-DCs are distinct from other DC subsets and have potential importance in spleen for immunity to blood-borne antigens. As production is maintained in cultures for >12 months, L-DC development evidently depends on self-renewing progenitors. To improve this culture system, highly purified HSCs were sorted from bone marrow and used to establish cocultures. Nonadherent cells produced were analyzed for surface marker expression and capacity to activate/inhibit T cells. Cocultures produced a pure population of L-DCs for up to 12 months, which were strong activators of CD8⁺ T cells. The in vitro production of a pure population of L-DCs from HSCs--in numbers amenable to in vitro assays of function and development--therefore represents an important advance.