Graft-versus-host disease (GVHD) is mediated by mature donor T cells contained in the hematopoietic stem cell graft. During the development of GVHD, signaling through a variety of costimulatory receptors plays an important role in allogeneic T cell responses. Even though delivery of costimulatory signals is a prerequisite for full activation of donor T cells in the phase of their interactions with host APCs, their involvement with GVHD might occur over multiple stages. Like many other aspects of GVHD, promise of therapeutic interventions with costimulatory pathways has been gleaned from preclinical models. In this review, I summarize some of the advances in roles of costimulatory molecules in GVHD pathophysiology and discuss preclinical approaches that warrant further exploration in the clinic, focusing on novel strategies to delete pathogenic T cells.