OX40 (CD134) is expressed on activated T cells; its ligand, OX40 ligand (OX40L) is expressed on dendritic cells, B cells, and activated endothelial cells. To determine how OX40-OX40L interaction affects graft-versus-host disease (GVHD), we used antagonistic anti-OX40L monoclonal antibody (mAb) or OX40^−/−donor or OX40L^−/− recipient mice. Similar degrees of GVHD reduction were observed with each approach. Despite the fact that OX40 is up-regulated on both CD4⁺ and CD8⁺ T cells isolated during GVHD, the major effects of OX40 ligation were on CD4⁺ and not CD8⁺ T-cell–mediated alloresponses as assessed in both GVHD and engraftment model systems. GVHD inhibition by blockade of the OX40/OX40L pathway did not require CD28 signaling. Some studies have indicated OX40 is essential for inducing T-helper type 2 (Th2) responses. However, in vivo blockade of OX40-OX40L interactions reduced GVHD mortality induced by either signal transducer and activator of transcription–6^−/− (Stat-6^−/−) (Th2-defective) or Stat-4^−/− (Th1-defective) major histocompatibility complex (MHC)–disparate splenocytes, indicating that the GVHD-ameliorating effects did not require Stat-4 or Stat-6 signaling. Although OX40L has been reported to be expressed on activated T cells, no effects on GVHD were observed when OX40L^−/− versus OX40L^+/+ T cells were infused in different models. These data provide insights as to the mechanisms responsible for OX40/OX40L regulation of GVHD.