Transplantation is the only cure for end-stage organ failure. Transplanted tissues are usually recognized by the immune system as foreign and are rapidly rejected in the absence of immunosuppression. Transplanted organs between genetically distinct individuals are termed allografts and their acute rejection is orchestrated by the activation of allospecific T cells. To prevent acute allograft rejection, current therapies suppress all T cells irrespective of their specificities and must be taken life-long, leaving patients with decreased defenses against infectious agents and cancers. The goal in transplantation research is to develop therapies with the capacity to induce graft-specific tolerance. Ideal therapies should be of short duration and target only alloreactive T cells, leaving other T cells competent to fight infections and cancers. Researchers have studied the mechanisms of activation/regulation of T cells in the hopes that manipulation of these pathways may facilitate the induction of tolerance. Activation of T cells requires recognition by the T cell receptor (TCR) of antigenic peptides presented within major histocompatibility complexes (MHC) on the surface of antigen-presenting cells (APCs). In addition, concurrent engagement of costimulatory receptors on T cells by ligands on APCs is also required for optimal T cell responses, such that the ultimate outcome of TCR engagement reflects the relative sum of multiple positive and negative costimulatory signals. Targeting costimulatory receptor/ligand pairs has been used effectively to induce allograft tolerance in specific rodent transplantation models. This strategy has however been less effective in larger mammals. In this review, we will summarize the different reagents used to target costimulatory molecules, their effects, and the possible reasons limiting their efficacy in higher order mammals.