Previous studies have shown that blockade of LIGHT, a T cell costimulatory molecule belonging to the TNF superfamily, by soluble lymphotoxin β receptor-Ig (LTβR-Ig) inhibits the cytotoxic T lymphocyte (CTL) response to host antigenic disparities and ameliorates lethal graft-versus-host disease (GVHD) in a B6 to BDF1 mouse model. Here, we demonstrate that infusion of an mAb against CD40 ligand (CD40L) further increases the efficacy of LTβR-Ig, leading to complete prevention of GVHD. We further demonstrate that alloantigen-specific CTLs become anergic upon rapid expansion, and persist in the tolerized mice asa result of costimulatory blockade. Transfer of anergic CTLs to secondary F1 mice fails to induce GVHD despite the fact that anergic CTLs can be stimulated to proliferate in vitro by antigens and cytokines. Our study provides a potential new approach for the prevention of lethal GVHD.