In this study, we addressed the role of tumor necrosis factor (TNF)-α and lymphotoxin (LT)-α in the development of colitis and defined the cellular sources (T cells versus non-T cells) of TNF (TNF-α and LT-α) relevant to disease development. After adoptive transfer of TNF^+/+ CD4⁺CD45RB^hi splenocytes into TNF^+/+ recombination activating gene (RAG)2^−/− mice, the recipients develop massive inflammation of the large intestinal mucosa concurrent with massive weight loss. In contrast, clinical signs of disease are completely absent in TNF^−/−RAG2^−/− recipients of TNF^−/− CD4⁺CD45RB^hi T cells, although elevated numbers of interferon-γ–producing cells are present in the colonic mucosa. Surprisingly, upon transfer of TNF^−/−CD4⁺CD45RB^hi T cells into TNF^+/+RAG2^−/− recipients, colitis develops with kinetics similar to those upon transfer of TNF^+/+CD4⁺CD45RB^hi donor cells. In contrast, no clinical signs of colitis are observed in TNF^−/−RAG2^−/− recipients of TNF^+/+CD4⁺CD45RB^hi T cells. This protection from colitis is not a consequence of the absence of LT-α, as TNF-α^−/−RAG2^−/− recipients of TNF-α^−/− CD4⁺CD45RB^hi T cells are also protected from colitis induction. These results demonstrate the importance of TNF production by non-T cells of the colonic mucosa in the pathogenesis of colitis and provide direct evidence for a nonredundant role of TNF-α in this mouse model of colitis.