We evaluated the safety and efficacy of donor lymphocyte infusion (DLI) with granulocyte colony-stimulating factor priming and short-term immunosuppressive agents for prophylaxis of relapse in patients with advanced leukemia after human leukocyte antigen (HLA)-mismatched T cell-replete hematopoietic stem cell transplantation (HCT). Twenty-nine patients received prophylactic DLI at a median 75 (33-120) days after HCT. Acute graft-vs-host disease (GVHD) grades 3-4 occurred in six patients, and all cases were controlled. Eleven patients were alive and relapse-free with a probability of leukemia-free survival (LFS) of 37.3 +/- 9.6% at 3 years. Chronic GVHD was associated with a lower relapse rate and higher probability of LFS. Prophylactic-modified DLI is feasible in patients with advanced leukemia to prevent relapse after HLA-mismatched HCT.