The expression of checkpoint blockade molecules PD-1, PD-L1, CTLA-4, and foxp3+CD25+CD4+ T cells (Tregs) regulate donor T cell activation and graft-vs-host disease (GvHD) in allogeneic hematopoietic stem cell transplant (allo-HSCT). Detailed kinetics of PD-1-, CTLA-4-, and PD-L1 expression on donor and host cells in GvHD target organs have not been well studied. Using an established GvHD model of allo-HSCT (B6 → CB6F1), we noted transient increases of PD-1- and CTLA-4-expressing donor CD4+ and CD8+ T cells on day 10 post transplant in spleens of allo-HSCT recipients compared with syngeneic HSCT (syn-HSCT) recipients. In contrast, expression of PD-1- and CTLA-4 on donor T cells was persistently increased in bone marrow (BM) of allo-HSCT recipients compared with syn-HSCT recipients. Similar differential patterns of donor T cell immune response were observed in a minor histocompatibility (miHA) mismatched transplant model of GvHD. Despite higher PD-1 and CTLA-4 expression in BM, numbers of foxp3+ T cells and Tregs were much lower in allo-HSCT recipients compared with syn-HSCT recipients. PD-L1-expressing host cells were markedly decreased concomitant with elimination of residual host hematopoietic elements in spleens of allo-HSCT recipients. Allo-HSCT recipients lacking PD-L1 rapidly developed increased serum inflammatory cytokines and lethal acute GvHD compared with wild-type (WT) B6 allo-HSCT recipients. These data suggest that increased expression of checkpoint blockade molecules PD-1 and CTLA-4 on donor T cells is not sufficient to prevent GvHD, and that cooperation between checkpoint blockade signaling by host cells and donor Tregs is necessary to limit GvHD in allo-HSCT recipients.