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Background Cell therapies in immuno-oncology have been a critical field in recent years, with natural killer (NK) cells being a promising and potent solution due to its pivotal role in the anti-inflammatory response and tumor surveillance. NK cells are well-suited for treating solid tumors, where they can penetrate the tumor matrix environment, activate fellow immune cells, and lyse cancer cells. Despite its potential, there is only a sparse following of NK-based cell therapies in clinical trials. This can be attributed to the challenges in sourcing and/or manufacturing high-quality NK cells. Representing 10 to 15% of the circulating lymphocytes in blood, NK cells are notoriously hard to obtain in sufficient numbers. NK cell lines were revealed to lack known IgG and IgM receptors which has resulted in NK-based cell therapies to use alternative sources such as pluripotent stem cells (PSCs). However, stem cell differentiation methods are extremely susceptible to variability which can easily alter indicators such as expansion rate, cell number, purity, and more. Furthermore, when considering the process from a clinical perspective, traditional culturing methods utilizing feeder cells are unfeasible due to safety and contaminant issues they pose as a secondary biological substance.Methods This study was designed to develop a serum-free, scalable, in vitro feeder-free system using PSCs for NK cell manufacturing.Results By optimizing and utilizing GMP-grade reagents throughout the entire manufacturing process, an 96.33% proportion of CD3- CD56+ NK cells were achieved in 25 days. At each stage, quality control assays were performed to validate and optimize the method before evaluating final NK cells on their biological function and cytotoxicity. As a serum and feeder-free system, this proposed method can be easily upscaled and outlines the framework towards the development of a closed, automated and GMP-compliant scalable expansion system for NK cell-based immunotherapy products.Conclusions This study demonstrates a scalable method for manufacturing iNK cells from iPSCs, oering a consistent and eicient approach to meet clinical demand. By optimizing protocols, a yield of 96.33% of iNK cells was achieved by Day 35. At day 49, expansion reached over 70K fold with greater than 90% viability with strong cytotoxic activity. This platform addresses limitations in NK cell availability and variability, providing a reliable source for o-the-shelf immunotherapies and supporting future clinical applications and broader accessibility in cell-based immunotherapy.