The recently described tumor necrosis factor (TNF) family member LIGHT (herpes virus entry mediator [HVEM]-L/TNFSF14), a ligand for the lymphotoxin (LT)β receptor, HVEM, and DcR3, was inactivated in the mouse. In contrast to mice deficient in any other member of the LT core family, LIGHT^−/− mice develop intact lymphoid organs. Interestingly, a lower percentage of LIGHT^−/−LTβ^−/− animals contain mesenteric lymph nodes as compared with LTβ^−/− mice, whereas the splenic microarchitecture of LIGHT^−/−LTβ^−/− and LTβ^−/− mice shows a comparable state of disruption. This suggests the existance of an additional undiscovered ligand for the LTβ receptor (LTβR) or a weak LTα₃–LTβR interaction in vivo involved in the formation of secondary lymphoid organs. LIGHT acts synergistically with CD28 in skin allograft rejection in vivo. The underlying mechanism was identified in in vitro allogeneic MLR studies, showing a reduced cytotoxic T lymphocyte activity and cytokine production. Detailed analyses revealed that proliferative responses specifically of CD8⁺ T cells are impaired and interleukin 2 secretion of CD4⁺ T cells is defective in the absence of LIGHT. Furthermore, a reduced ³[H]-thymidine incorporation after T cell receptor stimulation was observed. This for the first time provides in vivo evidence for a cooperative role for LIGHT and LTβ in lymphoid organogenesis and indicates important costimulatory functions for LIGHT in T cell activation.