Although several cytokines, including tumor necrosis factor (TNF), can promote the growth of dendritic cells (DCs) in vitro, the cytokines that naturally regulate DC development and function in vivo have not been well defined. Here, we report that membrane lymphotoxin (LT), instead of TNF, regulates the migration of DCs in the spleen. LTα^−/− mice, lacking membrane LTα/β and LTα₃, show markedly reduced numbers of DCs in the spleen. Unlike wild-type mice and TNF^−/− mice that have densely clustered DCs in the T cell zone and around the marginal zone, splenic DCs in LTα^−/− mice are randomly distributed. The reduced number of DCs in lymphoid tissues of LTα^−/− mice is associated with an increased number of DCs in nonlymphoid tissues. The number of splenic DCs in LTα^−/− mice is restored when additional LT-expressing cells are provided. Blocking membrane LTα/β in wild-type mice markedly diminishes the accumulation of DCs in lymphoid tissues. These data suggest that membrane LT is an essential ligand for the presence of DCs in the spleen. Mice deficient in TNF receptor, which is the receptor for both soluble LTα₃ and TNF-α₃ trimers, have normal numbers of DCs. However, LTβR^−/− mice show reduced numbers of DCs, similar to the mice lacking membrane LT α/β. Taken together, these results support the notion that the signaling via LTβR by membrane LTα/β is required for the presence of DCs in lymphoid tissues.