Abstract Relapse remains the most important cause of mortality after allogeneic hematopoietic cell transplantation (allo-HCT) and little progress has been made in the past decades. Chimeric antigen receptors targeting CD19 (CD19-CARs) redirect T cell effector functions to eliminate CD19-expressing leukemia and lymphoma cells. We found in pre-clinical studies that allogeneic donor-derived CD19-CAR T cells can have significant anti-lymphoma activity with minimal graft- versus-host disease (GVHD), an observation that was confirmed by others in clinical trials. We found that this was due to exhaustion and deletion of CAR-T cells attributable to cumulative signaling through both the alloreactive TCR and the CAR. In addition, we have demonstrated the feasibility of deletion of the endogenous TCR and expression of a CD19-CAR in T cells using CRISPR technology, which could further reduce potential alloreactivity of these cells. We have also developed an in vitro culture system to generate universal third-party T cell precursors (preT) that can be engineered to express CD19-CAR post-thymically to avoid negative selection. Upon adoptive transfer in allo-HCT recipients, these engineered preT cells can mature in the host’s thymus and exert anti-malignancy activity without GVHD. In addition to anti-malignancy potential, these engineered preT cells can also enhance immune reconstitution. Based on these findings and the clinical efficacy of adoptively transferred third party anti-viral T cells in allo-HCT recipients, we hypothesize that infusion of third-party “off-the-shelf” mature or preT cells expressing CARs and TCRs targeted against tumor-associated antigens will promote anti-malignancy activity and enhance immune reconstitution in allo-HCT recipients with minimal or no GVHD. We propose pre- clinical studies with engineered third-party mature and preT cells to prevent or treat relapse after allo-HCT. In Aim 1, we will use pre-clinical allo-HCT models to evaluate the anti-malignancy activity, GVHD potential and persistence of third-party T cells whose endogenous TCR has been deleted using CRISPR and that express (1.1) CD19-CAR, (1.2) triple-antigen-specific CARs, or (1.3) CD19-CAR/WT-1 specific TCR. In Aim 2, we will study third-party preT cells expressing (2.1) a CD19-CAR, (2.2) a CD19-CAR and cytokines, or (2.3) CD19-CAR/WT1 specific TCR. We have already developed two allo-HCT clinical trials with donor-derived CD19-CAR T cells or third-party preT cells, which together with our extensive clinical experience with CAR T cells will facilitate the translation of the proposed preclinical studies to decrease relapse in allo-HCT patients with hematologic malignancies using third party CAR T cells.