Abstract Several autologous CAR-T cell therapies have been approved for the treatment of B cell lymphomas and leukemias and a number of trials are in progress to investigate the use of allogeneic, donor derived CAR-T and CAR-NK cell therapies for multiple tumor indications. However, sourcing cell donors, editing cells and generating large batches of clinical grade allogeneic CAR-T and CAR-NK can be challenging. To overcome these difficulties, we are developing NK and T cell-based therapies derived from induced pluripotent stem cells (iPSCs). iPSCs have the capacity to be genetically engineered, enabling precise edits that can enhance the function of cell therapies, and indefinitely expanded in culture, allowing for the production of highly uniform master cell banks. Yet, an open question of this system is the extent to which the donors’ genetic background shapes the phenotype of the iPSC-derived NK and T cells. To answer this question, we measured the genome-wide landscape of chromatin accessibility and gene expression in iNK cells generated from multiple donors. Donor background was linked to significant changes in chromatin accessibility (~30% of regions tested) and expression (~15% genes). We found accessibility changes at binding sites for transcription factors regulating various aspects of NK cell function, including differentiation (GATA2, EOMES, and ETS1), activation (AP-1, T-bet), and cytokine production (STAT3, STAT5), indicating that the donor background could impact the functionality of iPSC-derived NK cells. In particular, we noted that iNK cells derived from one donor, which had decreased effector functions compared to other cell lines, showed increased accessibility at BACH2 binding sites, a key negative regulator of NK cell function. iNK cells derived from this donor also showed an increase in expression of genes linked to NK cell differentiation and cell development, and a decrease in genes involved in cell cycle, cell proliferation, and inflammatory response pathways. Taken together, these data indicate the importance of assessing the impact of donor background on iPSC derived cell therapies through a variety of functional and genomic assays. Citation Format: Rohith Srivas, Snehal Nariya, Melissa Rojsza, Barry A. Morse, Ohad Manor, Rupesh Amin, Luis Borges. Extensive epigenetic and transcriptomic donor-specific differences observed in iPSC derived allogenic NK (iNK) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6083.