Several roadblocks preclude clinical translation of natural killer (NK) therapies including (1) lack of scalable platforms for clinical-scale manufacturing, (2) significant donor-to-donor variability, and (3) need for robust and predictive preclinical assays to validate NK efficacy. Umbilical cord blood (UCB) expanded hematopoietic stem cells (xHSCs) represent ideal sources for differentiating large numbers of multifunctional NK cells with a consistent phenotype and function, but xHSC-derived NK cells (xHSC-NKs) have not been thoroughly characterized in clinically-relevant contexts. Cryopreserved UCB HSCs were expanded >1000-fold in vitro using the patented small molecule IBR403, and differentiated into NK cells. Cells were screened using flow cytometry, xCELLigence, and advanced assays (e.g. serial rechallenge, organoid co-culture, and in vivo models). xHSC-NKs expressed high levels of canonical activating receptors including NKG2D, DNAM-1, NKp30, NKp44, and NKp46, and degranulated and produced inflammatory cytokines upon stimulation with target cells similarly to peripheral blood (pb) NK cells. xHSC-NKs featured high cytotoxicity against diverse tumor cell lines without the need for CAR engineering, allowing us to identify optimal donors for multiple clinical applications. Serial rechallenge assays showed NKs exhibited significant donor-derived variability in their responses to repeated tumor exposures. xHSC-NKs eradicated 3D patient-derived cancer organoids comparably to pbNKs. Finally, xHSC-NKs acquired a mature phenotype (KIR+CD16+) and exhibited long-term persistence and high killing capacity after 10 weeks in vivo. Our integrated screening and manufacturing pipeline not only solves a major gap in cell therapy development, but also allows us to answer fundamental questions about human NK differentiation and function. Overall, xHSC-NK cells demonstrate high efficacy against tumor cells and clear therapeutic potential, with our platform enabling scalable, cost-effective manufacturing and rigorous donor prescreening to produce thousands of potent allogeneic NK doses from a single UCB unit, democratizing access to durable therapies and improving patient outcomes.