We demonstrate that functional and phenotypic equivalents of mouse splenic CD8⁺ and CD8⁻ conventional dendritic cell (cDC) subsets can be generated in vitro when bone marrow is cultured with fms-like tyrosine kinase 3 (flt3) ligand. In addition to CD45RA^high plasmacytoid DC, two distinct CD24^high and CD11b^high cDC subsets were present, and these subsets showed equivalent properties to splenic CD8⁺ and CD8⁻ cDC, respectively, in the following: 1) surface expression of CD11b, CD24, and signal regulatory protein-α; 2) developmental dependence on, and mRNA expression of, IFN regulatory factor-8; 3) mRNA expression of TLRs and chemokine receptors; 4) production of IL-12 p40/70, IFN-α, MIP-1α, and RANTES in response to TLR ligands; 5) expression of cystatin C; and 6) cross-presentation of exogenous Ag to CD8 T cells. Furthermore, despite lacking surface CD8 expression, the CD24^high subset contained CD8 mRNA and up-regulated surface expression when transferred into mice. This culture system allows access to bona fide counterparts of the splenic DC subsets.