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Abstract Delayed immune reconstitution following allogeneic hematopoietic cell transplant (allo-HCT) increases the risk of infection, graft failure, and relapse. To address this, immobilized DLL4-Fc (recombinant Delta-like 4 fused to an Fc domain) has been developed to generate human T lymphoid precursors from hematopoietic stem and progenitor cells (HSPCs) in a scalable, GMP-compatible manner. While this platform is under evaluation in early-phase clinical trials, its mechanistic and therapeutic potential has not been systematically tested in preclinical allo-HCT models. To enable preclinical evaluation, we adapted and optimized the DLL4-Fc platform for murine HSPCs. By culturing bone marrow (BM)-derived Lineage-Sca1+cKit+ (“LSK”) cells with IL-7, Flt3L, and SCF on DLL4-Fc coated plates, we consistently achieved >2,000-fold expansion over 2–3 weeks. The resulting population consisted of >95% purity for the DN2 (lineage-CD44+CD25-) and DN3 (lineage-CD44+CD25-) T cell precursors (hereafter “PreT”), providing a robust and scalable source of murine PreTs for downstream in vivo applications. To assess in vivo functionality, we used a well-established MHC-disparate murine allo-HCT model (C57BL/6→BALB/c) with LSK allograft. Adoptively transferred DLL4-generated PreTs successfully engrafted in the thymus and significantly increased total thymic cellularity compared to controls (p<0.05). This effect was dose-dependent, beginning at 2×10⁶ cells (p<0.05) and further enhanced at 4×10⁶ cells (p<0.01), supporting a quantitative benefit of PreT dosing. PreTs progressed through canonical thymocyte stages by day 30 post-transplant, confirming successful intrathymic maturation. Beyond their intrathymic development, PreTs also promoted regeneration of the thymic microenvironment. On day 30 post-transplant, we observed accelerated recovery of thymic stromal compartments, including thymic epithelial cells (TECs, p<0.01) and endothelial cells (ECs, p<0.05), accompanied by increased Ki-67 expression (both p<0.05), indicating enhanced proliferation. Notably, PreT therapy significantly increased de novo thymopoiesis from donor LSK cells, beginning at the early thymic progenitor stage and spanning all maturation stages. By day 30, we observed increased numbers of mature CD4+ and CD8+ single-positive T cells, derived from both PreTs and donor HSPCs (all p<0.05), in the thymus and spleen, indicating successful intrathymic maturation and peripheral emigration. Through their dual impact on both direct thymopoiesis and stromal regeneration, PreTs significantly enhanced overall thymic cellularity on day 30 (p<0.0001). In contrast, TCRb-/- PreTs failed to accelerate TEC or EC recovery, suggesting that the effect on stromal regeneration is dependent on T cell lineage commitment. Transcriptomic analysis using bulk RNA sequencing revealed that cultured PreTs, compared to LSKs, expressed higher levels of thymotropic factors known to support stromal regeneration, including Tnfsf11, Fgf7, Bmp4, Lta. Further mechanistic studies are ongoing to define the Pre-stroma crosstalk that supports thymic regeneration. We next assessed the effects of PreT therapy in allo-HCT recipients with heightened thymic impairment due to aging or post-transplant cyclophosphamide (PTCy). In these clinically relevant settings, PreT treatment significantly increased total thymic cellularity in both 5- and 9-month-old recipients, as well as in mice receiving PTCy (50mg/kg on days +3/+4) (all p<0.05). These findings demonstrate that PreT therapy remains effective despite age-related stromal degeneration and direct exposure to cyclophosphamide, supporting its broader utility in allo-HCT. We evaluated whether PreT therapy enhances peripheral T cell function by infecting allo-HCT recipients with acute lymphocytic choriomeningitis virus (Armstrong strain) on day 30 post-transplant. By day 8 post-infection, PreT-treated mice showed significantly higher frequencies of CD8+ T cells producing TNF-α, IFN-γ, and GzmB, indicating enhanced antiviral responses. We then assessed graft-versus-tumor activity across multiple relapse models (lymphoma, plasmacytoma, leukemia) and found that PreT therapy consistently reduced tumor burden regardless of MHC compatibility. Importantly, no graft-versus-host disease was observed. These findings highlight DLL4-derived PreTs as a GMP-compatible cellular therapy to enhance immune reconstitution and confer antiviral and antitumor activity post-HCT.