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Background Natural killer (NK) cells offer a promising allogeneic immunotherapy for various malignancies due to their innate ability to target tumor cells via a balance of activating and inhibitory receptors. This mechanism enables effective antitumor activity with minimal toxicity. However, clinical translation has been hindered by challenges including: limited scalability of cell sources for manufacturing, significant donor-to-donor variability, and the need for robust, predictive preclinical assays.Methods We developed a scalable NK cell manufacturing and screening platform leveraging IBR403, a proprietary small molecule, to expand hematopoietic stem cells (HSCs) from small-volume cord blood segments (≤300 µL) by 1,000- to 1,000,000-fold, while preserving the main 25 mL cord blood unit (CBU) for selective large-scale production. Expanded HSCs (xHSCs) were differentiated into NK cells (xHSC-NKs) and functionally characterized using flow cytometry, xCELLigence assays, and advanced models including serial tumor rechallenge, 3D organoid co-culture, and in vivo studies.Results xHSC-NKs from optimal donors expressed high levels of activating receptors (e.g., NKG2D, DNAM-1), degranulated efficiently, and secreted proinflammatory cytokines upon stimulation—mirroring peripheral blood NK (pbNK) function. These xHSC-NKs exhibited potent cytotoxicity across diverse tumor cell lines, enabling identification of high-performing donor profiles across various hematological and solid tumor types. In serial rechallenge assays, xHSC-NKs displayed variable responses based on donor origin, highlighting the importance of functional prescreening. xHSC-NKS also eliminated patient-derived 3D tumor organoids comparably to pbNKs. Additionally, xHSC-NKs acquired a mature phenotype (KIR+CD16+) and exhibited long-term persistence and high killing capacity after 10 weeks in vivo.Conclusions Our platform enables robust expansion and differentiation of CBU-derived HSCs into highly functional NK cells, supporting scalable, cost-effective production. With integrated prescreening and long-term in vivo efficacy, xHSC-NKs represent a powerful allogeneic cell therapy platform with the potential to deliver durable antitumor responses and broaden patient access to NK-based immunotherapies.