In this project we aimed to search for clinical relevant minor histocompatibility antigens (MiHAs) by combining HLA-ligandome analyses with RNA expression databases. T cells recognizing these clinically relevant MiHAs could potentially induce a leukemia specific immune reaction after allogeneic stem cell transplantation (allo-SCT) without the detrimental induction of graft versus host disease. Using this strategy, 34 MiHA peptide candidates restricted to different HLA-types were identified. To validate the potential clinical relevance of the MiHA candidates T cells specific for these MiHA candidates were isolated from healthy donors by large-scale HLA-tetramer enrichments combined with single cell expansion and high-throughput functional analyses. We isolated peptide specific T-cells against several newly identified MiHAs, which recognized endogenously processed and presented peptide as was assessed in stimulation assays using tandem minigene constructs, indicating potent reactivity of the MiHA specific T cell clones. The T cells directed against those leukemia-specific MiHAs could potentially be valuable for cell therapy purposes for the treatment of acute leukemia and high-grade hematological tumors.