Deficient immunoregulation by CD4⁺ T cells is an important susceptibility trait for inflammatory bowel disease, but the role of other regulatory cell types is less understood. This study addresses the role and mechanistic interaction of B cells and CD8⁺ T cells in controlling immune-mediated colitis. The genetic requirements for B cells and CD8⁺ T cells to confer protective immunoregulation were assessed by co-transfer with colitigenic Gi2^-/- T cells into immune deficient mice. Disease activity in Gi2^-/- T cell recipients was evaluated by CD4⁺ T intestinal lymphocyte abundance, cytokine production levels, and large intestine histology. B cells deficient in B7.1/B7.2, CD40, MHC II (Abb), or native BCR (MD4) were competent for colitis protection. However, TAP-1-deficient B cells failed to protect, indicating a requirement for peptide MHC I presentation to CD8⁺ T cells. CD8⁺ T cells deficient in native TCR repertoire (OT-1) or cytolysis (perforin-/-) also were non-protective. These finding reveal an integrated role for antigen-specific perforin-dependent CD8⁺ T cell cytotoxicity in colitis immunoregulatory, and its efficient induction by a subset of mesenteric B lymphocytes.