Abstract Acute graft-versus-host disease (aGVHD) hinders the efficacy of allogeneic hematopoietic cell transplantation (HCT). Plasma levels of soluble membrane-bound ST2 (ST2) are elevated in human and murine aGVHD and correlated to type 1 T cells response. ST2 signals through the adapter protein MyD88. The role of MyD88 in T cells during aGVHD has yet to be elucidated. We found that knocking out MyD88 in the donor T cells protected against aGVHD independent of IL-1R and TLR4 signaling in two murine HCT models. This protection was entirely driven by MyD88−/− CD4 T cells. Transplanting donor MyD88−/− conventional T cells (Tcons) with wild-type (WT) or MyD88−/− regulatory T cells (Tregs) lowered aGVHD severity and mortality. Transcriptome analysis of sorted MyD88−/− CD4 T cells from the intestine 10 d post-HCT showed lower levels of Il1rl1 (gene of ST2), Ifng, Csf2, Stat5, Batf, and Jak2. Transplanting donor ST2−/− Tcons with WT or ST2−/− Tregs showed a similar phenotype with what we observed when using donor MyD88−/− Tcons. Decreased ST2 was confirmed at the protein level with less secretion of soluble ST2 and more expression of ST2 compared with WT T cells. Our data suggest that Treg suppression from lack of MyD88 signaling in donor Tcons during alloreactivity uses the ST2 but not the IL-1R or TLR4 pathways, and ST2 represents a potential aGVHD therapeutic target sparing Tregs.