Proper lymph node (LN) development requires tumor necrosis factor-related activation-induced cytokine (TRANCE) expression. Here we demonstrate that the defective LN development in TRANCE(-/)- mice correlates with a significant reduction in lymphotoxin (LT)alphabeta(+)alpha(4)beta(7)(+)CD45(+)CD4(+)CD3(-) cells and their failure to form clusters in rudimentary mesenteric LNs. Transgenic TRANCE overexpression in TRANCE(-/)- mice results in selective restoration of this cell population into clusters, and results in full LN development. Transgenic TRANCE-mediated restoration of LN development requires LTalphabeta expression on CD45(+) CD4(+)CD3(-) cells, as LNs could not be induced in LTalpha(-/)- mice. LTalpha(-/)- mice also showed defects in the fate of CD45(+)CD4(+)CD3(-) cells similar to TRANCE(-/)- mice. Thus, we propose that both TRANCE and LTalphabeta regulate the colonization and cluster formation by CD45(+) CD4(+)CD3(-) cells in developing LNs, the degree of which appears to correlate with the state of LN organogenesis.