To elucidate the capacity of murine early hematopoietic progenitor cells (HPCs) to differentiate into dendritic cells (DCs), lineage phenotypes (Lin)⁻c-kit⁺ HPCs were highly purified from either wild-type or tumor necrosis factor (TNF) receptor p55 (TNF-Rp55)-deficient mice. Upon culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF) for 14 days, wild-type mouse Lin⁻c-kit⁺ HPCs did not exhibit characteristic features of DC such as sheet-like projections and veil processes. Moreover, these cells expressed a marginal level of DC markers such as DEC-205, CD86, and barely supported allogenic MLR. However, the addition of mouse TNFα generated a large number of cells with typical DC morphology, expression of high levels of Ia, DEC-205, CD86, and function of stimulating allogenic MLR. Moreover, a proportion of these mature DCs and thymic DCs expressed Thy-1 mRNA as well as Thy-1 antigen, whereas freshly isolated splenic DCs did not. These results suggested that DCs generated in our culture system phenotypically resemble thymic ones. In contrast, mouse TNFα failed to induce TNF-Rp55-deficient mice-derived Lin⁻c-kit⁺ HPCs to generate DCs with characteristic morphology, immunophenotype, and accessory function for T cells under the same culture conditions, suggesting a crucial role of TNF-Rp55 in TNFα-mediated DC differentiation from HPCs. Interestingly, human TNFα, which can bind to mouse TNF-Rp55 but not TNF-Rp75, was incapable to augment DC generation from wild-type mouse Lin⁻c-kit⁺ HPCs. Collectively, these results suggest that TNFα has a pivotal role in DC generation from murine early HPCs in collaboration with GM-CSF and SCF through the interaction of TNF-Rp55 and TNF-Rp75.