Despite therapeutic advancements, GVHD is a major complication of HSCT. In current models of GVHD, tissue injury induced by cytotoxic conditioning regimens, along with translocation of microbes expressing Pathogen Associated Molecular Patterns (PAMPs), result in activation of host antigen-presenting cells (APC) to stimulate alloreactive donor T lymphocytes. Recent studies have demonstrated that in many pathologic states, tissue injury results in the release of mitochondria from the cytoplasm to the extracellular space. We hypothesized that extracellular mitochondria, which are related to archaebacteria, could also trigger GVHD by stimulation of host APC. We found that clinically relevant doses of radiation or busulfan induced extracellular release of mitochondria by various cell types, including cultured intestinal epithelial cells. Conditioning-mediated mitochondrial release was associated with mitochondrial damage and impaired quality control but did not affect the viability of the cells. Extracellular mitochondria directly stimulated host APCs to express higher levels of MHC-II, co-stimulatory CD86, and pro-inflammatory cytokines, resulting in increased donor T cell activation, and proliferation in mixed lymphocyte reactions. Analyses of plasma from both experimental mice and a cohort of children undergoing HSCT demonstrated that conditioning induced extracellular mitochondrial release in vivo. In mice undergoing MHC mismatched HSCT, administration of purified syngeneic extracellular mitochondria increased host APC activation and exacerbated GVHD. Our data suggests that pre-HSCT conditioning results in extracellular release of damaged mitochondria which increase alloreactivity and exacerbate GVHD. Therefore, decreasing the extracellular release of damaged mitochondria following conditioning could serve as a novel strategy for GVHD prevention.