Hematopoietic cell lineages are best described in terms of distinct progenitors with limited differentiative capacity. To distinguish cell lineages, it is necessary to define progenitors and induce their differentiation in vitro. We previously reported in vitro development of immature dendritic-like cells (DCs) in long-term cultures (LTCs) of murine spleen, and in cocultures of spleen or bone marrow (BM) over splenic endothelial cell lines derived from LTCs. Cells produced are phenotypically distinct CD11b(hi)CD11c(lo)CD8(-)MHC-II(-) cells, tentatively named L-DCs. Here we delineate L-DC progenitors as different from known DC progenitors in BM and DC precursors in spleen. The progenitor is contained within the lineage-negative (Lin)(-)c-kit(+) subset in neonatal and adult spleen. This subset has multipotential reconstituting ability in mice. In neonatal spleen, the progenitor is further enriched within the c-kit(lo) and CD34(+) subsets of Lin(-)c-kit(+) cells. These cells seed cocultures of splenic endothelial cells, differentiating to give L-DCs that can activate T cells. L-DC progenitors are distinguishable from described splenic CD11c(lo) DC precursors and from Fms-like tyrosine kinase 3(+) DC progenitors in BM. Overall, this study confirms that LTCs are a physiologically relevant culture system for in vitro development of a novel DC type from spleen progenitors.