The study of early events in dendritic cell (DC) differentiation is hampered by the lack of homogeneous primary cell systems that allow the study of cytokine-driven, transitional DC differentiation steps. The CD34(+) acute myeloid leukemia cell line MUTZ-3 displays a unique ability to differentiate into interstitial DC (IDC) and Langerhans cells (LC) in a cytokine-dependent manner. Phenotypic characterization revealed MUTZ-3 to consist of three distinct subpopulations. Small CD34(+)CD14(-)CD11b(-) progenitors constitute the proliferative compartment of the cell line with the ability to differentiate through a CD34(-)CD14(-)CD11b(+) stage to ultimately give rise to a morphologically large, nonproliferating CD14(+)CD11b(hi) progeny. These CD14(+)CD11b(hi) cells were identified as common, immediate myeloid DC precursors with the ability to differentiate into LC and IDC, exhibiting characteristic and mutually exclusive expression of Langerin and DC-specific ICAM-grabbing nonintegrin, respectively. The identity of the MUTZ-3-derived LC subset was confirmed further by the presence of Birbeck granules. We conclude that the MUTZ-3 cell line provides a ready and continuous supply of common myeloid precursors, which should facilitate further study of the ontogeny of myeloid DC lineages.