Inhibitors of apoptosis proteins (IAPs) regulate apoptosis, but little is known about the role of IAPs in the regulation of immunity. Development of IAP inhibition by second mitochondria-derived activator of caspase (SMAC) mimetics is emerging as a novel therapeutic strategy to treat malignancies. We explored the role of IAPs in allogeneic immunity with 2 distinct yet complementary strategies, namely, chemical and genetic approaches, in clinically relevant models of experimental bone marrow transplantation (BMT). The small-molecule pan-IAP inhibitor SMAC mimetic AT-406 aggravated gastrointestinal graft-versus-host disease (GVHD) in multiple models. The role of specific IAPs in various host and donor cellular compartments was explored by utilizing X-linked IAP (XIAP)- and cellular IAP (cIAP)-deficient animals as donors or recipients. Donor T cells from C57BL/6 cIAP1-/- or XIAP-/- animals demonstrated equivalent GVHD severity and allogeneic responses, both in vivo and in vitro, when compared with B6 wild-type (B6-WT) T cells. By contrast, when used as recipient animals, both XIAP-/- and cIAP1-/- animals demonstrated increased mortality from GVHD when compared with B6-WT animals. BM chimera studies revealed that cIAP and XIAP deficiency in host nonhematopoietic target cells, but not in host hematopoietic-derived cells, is critical for exacerbation of GVHD. Intestinal epithelial cells from IAP-deficient animals showed reduced levels of antiapoptotic proteins as well as autophagy-related protein LC3 after allogeneic BMT. Collectively, our data highlight a novel immune cell-independent but target tissue-intrinsic role for IAPs in the regulation of gastrointestinal damage from GVHD.