ABSTRACT To establish a transfusion-associated graft-versus-host disease (TA-GVHD) mouse model with busulfan and fludarabine for effective treatment evaluation. BALB/c (H-2d) mice were injected with busulfan (15 mg/kg) and fludarabine (30 mg/kg) twice a day for 4 days. The mice were transfused with 106 T cell-depleted bone marrow (TCD-BM )and cells in different groups 3 days after chemotherapy: syngeneic BALB/c, MHC minor mismatch DBA/2 (H-2d), or MHC major mismatch C57BL/6(H2-b). Recipient BALB/c mice were injected with either blood only or blood+splenocyte. TA-GVHD was monitored in terms of body weight loss, clinical scores, and survival. Dexamethasone (50 mg/kg), cyclophosphamide (50 mg/kg), cyclosporine A (30 mg/kg), and anti-CD3 (1 mg/kg) were injected to each group to examine the treatments. Blood transfusion alone is insufficient to induce TA-GVHD in a chemotherapy-based mouse model. A MHC-mismatched TA-GVHD model can be induced by splenocyte and blood transfusion. This MHC-mismatched TA-GVHD model was resistant to dexamethasone treatment. Treatment based on anti-CD3 monoclonal antibody slightly ameliorated TA-GVHD. Treatment effectiveness was associated with T-cell depletion following activation by anti-CD3. Busulfan and fludarabine chemotherapy regimen can be used to establish a TA-GVHD mouse model. Anti-CD3 monoclonal antibody is a potential alternative to treat TA-GVHD.