Objective To explore the expression of costimulators in mice after allogenetic hematopoietic stem cell transplantation and its relationship with acute graft versus host disease (aGVHD). Methods Five C57BL/6J (H2KD^-H2KB⁺) male mice were selected as donors. Thirty CB6F1 (H2KD⁺H2KB⁺) female mice were selected as recipients and randomized into three groups:total body irradiation (TBI), bone marrow transplantation (BM) and aGVHD groups, with 10 mice in each group. The mice in the TBI group received radiation only without injecting any cells. The mice in the BM group were injected with 5×10⁶ bone marrow cells without T lymphocytes from donors after radiation. The mice in the aGVHD group were simultaneously injected with 5×10⁶ bone marrow cells without T lymphocytes and 3×10⁷ spleen cells from donors after radiation. The survival rates of mice in the three groups were evaluated using log-rank survival curve. The expression levels of costimulators, including cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), programmed death 1 (PD-1), inducible costimulator (ICOS) and CD28, on CD4⁺ and CD8⁺ T lymphocytes, were detected using flow cytometry at 7, 14, 21 and 28 d after transplantation. At 21 d after transplantation, the expression levels of interleukin (IL)-17, interferon γ (IFN-γ) and IL-4 in CD4⁺ T lymphocytes, and the costimulator ligands in the colon tissues, were detected with flow cytometry and 3,3'-diaminobenzidine (DAB) staining, respectively. Results The mice in the TBI group all died within 19 d after irradiation. The mice in the BM group survived 30 d after irradiation without aGVHD. The median onset time of aGVHD symptoms and survival of mice in the aGVHD group were 14 (11-18) d and 22 (13-30) d, respectively. The expression levels of CTLA-4 and PD-1 on CD4⁺ and CD8⁺ T lymphocytes were decreased after transplantation, while the expression levels of ICOS and CD28 were on the rise. At 28 d after transplantation, the expression levels of CTLA-4 and PD-1 on CD4⁺ and CD8⁺ T lymphocytes were significantly lower in the aGVHD group than those in the BM group (all P<0.05), and the expression levels of ICOS and CD28 were significantly higher (all P<0.05). DAB staining showed that CD80, ICOS ligand (ICOSL), and PD-1 ligand (PD-L1) were negative in the colon tissues of mice in the BM group, and the positive expression rates of CD80, ICOSL, and PD-L1 in the aGVHD group were 40%, 80% and 80%, respectively. Compared with the BM group, the expression levels of IL-17 and IFN-γ in CD4⁺ T lymphocytes in the aGVHD group were significantly increased, while the expression level of IL-4 was significantly decreased (all P<0.05). Conclusion In aGVHD mice after allogeneic hematopoietic stem cell transplantation, the expression of costimulator CTLA-4 is gradually decreased over time. CTLA-4, ICOS and PD-1 may participate in the development and progression of aGVHD by regulating the distribution of T lymphocyte subgroups such as T-helper cell (Th)1, Th2 and Th17.