Acute graft-versus-host disease (aGVHD) is an important complication of allogeneic hematopoietic cell transplantation (HCT). The majority of aGVHD mouse models are based on radiation conditioning and bone marrow as graft, despite that most allo-HCTs performed now in clinic are based on chemotherapy conditioning and G-CSF mobilized graft. Aiming for a higher translational value, we constructed an MHC major mismatched [C57BL/6 (H-2 Kb) to BALB/c (H-2Kd)] aGVHD mouse model based on busulfan/cyclophosphomide (BU–CY) conditioning and human G-CSF mobilized splenocytes as graft. Allogeneic transplanted mice showed quick and profound donor engraftment. Moreover, there were quick onset (day +7) of typical clinical and histopathological signs of aGVHD, which gradually developed to extensive aGVHD. In addition, CD8+ T cells were the main aGVHD contributing T-cell subtype. No toxicity or GVHD signs were observed in the syngeneic setting. This clinical-relevant model offers a promising platform for future studies on aGVHD.