Abstract Dendritic cells (DCs) generated from bone marrow (BM) precursor cells of C57BL/6 (B6.WT) mice and cultured in the presence of granulocyte macrophage-colony stimulating factor differentiate to mature BM-DCs spontaneously. These mature DCs are characterized by high levels of major histocompatibility complex (MHC) class II, CD40, and CD86 on their surface. To analyze the involvement of tumor necrosis factor (TNF) and the related cytokine lymphotoxin (LT)α in DC maturation, we studied the development of DCs from the BM of B6.TNF−/−, B6.LTα−/−, and B6.TNF/LTα−/− mice and compared it to B6.WT mice. Although the development of BM precursor cells to the level of immature DCs (CD11c+, MHC class IIlow, CD40low, and CD86low) was equivalent in all genotypes, B6.TNF−/− and B6.TNF/LTα−/− cells showed an impaired capacity to differentiate to mature DCs. In contrast, mature BM-DCs generated from LTα-negative, immature DCs developed like B6.WT cells. Further studies revealed that once matured, the phenotype of all tested genotypes was comparable. They expressed high levels of CD40 and CD86, were exclusively positive for the chemokine receptor (CCR)7 but negative for CCR5 and CCR2, and were able to enter the paracortex of draining lymph nodes. The limited maturation of TNF-deficient BM-DCs could be restored by mixing TNF-negative with TNF-positive Ly5.1 BM cells, and maturation of B6.WT DCs could be blocked with an anti-TNF monoclonal antibody. The substitution of B6.TNF−/− BM cells with recombinant TNF revealed promotion or suppression of BM-DC maturation depending on the point of time of TNF addition.