Allo-HSCT is a curative therapy for hematologic malignancies owing to GvL effect mediated by alloreactive T cells; however, the same T cells also mediate GvHD, a severe side effect limiting the widespread application of allo-HSCT in clinics. Invariant natural killer T (iNKT) cells can ameliorate GvHD while preserving GvL effect, but the clinical application of these cells is restricted by their scarcity. Here, we report the successful generation of third-party HSC-engineered human iNKT (HSC-iNKT) cells using a method combining HSC gene engineering and HSC differentiation. The HSC-iNKT cells closely resembled the CD4CD8 subsets of endogenous human iNKT cells in phenotype and functionality. These cells displayed potent anti-GvHD functions by eliminating antigen-presenting myeloid cells and in xenograft models without negatively impacting tumor eradication by allogeneic T cells in preclinical models of lymphoma and leukemia, supporting HSC-iNKT cells as a promising off-the-shelf cell therapy candidate for GvHD prophylaxis.