A major limitation in current allogeneic hematopoietic stem cell transplantation (alloHSCT) is disease relapse after transplant, indicating that donor-derived T cells are inadequate in imparting an effective antitumor response. The current standard treatment approach to relapse utilizes donor lymphocyte infusions that have limited documented efficacy and are also associated with significant morbidity mainly related to graft-versus-host disease. We have previously shown that marrow-infiltrating lymphocytes (MILs) have a broader antigenic specificity compared with their peripheral blood counterpart in an autologous adoptive T-cell therapy setting. Here, we extend these observations to examine the ability of MILs obtained from patients after an alloHSCT to generate measurable tumor-specific immunity. We show here that allogeneic donor-derived marrow-infiltrating lymphocytes (ddMILs) obtained from patients who underwent alloHSCT with posttransplant cyclophosphamide could be reproducibly expanded and activated with anti-CD3/CD28 beads. Phenotypic characterization of ddMILs subpopulations revealed the prevalence of a central memory phenotype. Polyclonally activated ddMILs displayed measurable in vitro antitumor activity. Furthermore, activated ddMILs from all patients effectively targeted third-party allogeneic antigens, but showed no reactivity toward self-antigens presented in an HLA-restricted manner. Collectively, these results underscore the intrinsic polyclonal tumor-specificity of activated ddMILs and describe a novel approach for the generation of tumor-specific T cells that are suitable for adoptive immunotherapy of hematological malignancies relapsed after alloHSCT. This approach has a potential to significantly increase the tumor-specificity and reduce the toxicities associated with current standard donor lymphocyte infusion approaches.