Objective To explore the effect of costimulatory moleculars expressed peripheral CD4+ T lymphocyts on graft-versus-host disease(GVHD) after allogeneic hematopoietic stem cell transplantation (alloHSCT). Methods 21 patients who suffered of hematology diseases or malignant solid tumors were underwent allo-HSCT and 10 normal individuals were enrolled in the study. The levels of CD28, CD80 and CD152expressions on peripheral CD4+ T lymphocytes were detected by FCM in different time(before allo-HSCT, 7 day,14 day, 21 day, 30 day after allo-HSCT, thetime of GVHD and the time after GVHD treated). STR-PCR was used to detect micro-satellites chimeras forming. Results All 21 patients achieved engraftment. By STR-PCR assay, 12 cases formed complete chimeras (CC) and 9 cases formed mixed chimeras (MC). A multivariate COX survival function modle analysis showed: CD4 CD152 was independent prognostic factors for GVHD (x2=13.128,P ＜0.0001). Patients with GVHD demonstrated higher CD4+ CD28+, CD4+ CD80+ and CD4+ CD152+ T cell levels than those without GVHD (P ＜0.01); patients with aGVHD demonstrated higher than those with cGVHD (P ＜0.05)and without GVHD (P ＜0.05); Patients with GVHD demonstrated lower CD4+ CD152+ T cell level than those without GVHD (P ＜0.01); the same result occured between aGVHD and cGVHD and without GVHD.Conclusion The incidence of GVHD between NST and traditional HSCT have no difference. B7-CD28/CD152 costimulatory pathway plays a critical role in the development of GVHD.