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Background Researchers have increasingly explored the potential of adoptive transfer of expanded NK cells as a therapeutic approach for cancer treatment. Culturing NK cells with irradiated Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (LCL) in the presence of IL-2 leads to extensive NK cell proliferation. Leveraging this insight, we developed a GMP protocol to expand NK cell populations for infusion into cancer patients in a clinical trial. Despite these advances, there remains a significant gap in published research characterizing the molecular interactions responsible for the dramatic proliferation of NK cells induced by EBV-transformed B-cells.Methods LCL were profiled by RNA-sequencing, with and without irradiation, to measure expression of molecules known to influence NK cell behavior. Proliferation of primary human NK cells was assessed in the presence of LCL and IL-2 along with monoclonal antibodies that block candidate interactions. Findings were confirmed by CRISPR Cas9-mediated gene editing of LCL to disrupt NK cell ligands and subsequent co-culture assays of NK cell proliferation.Results These experiments identified a primary role of 4-1BBL, which is augmented on LCL by irradiation, interacting with 4-1BB on NK cells to trigger entry into cell cycle. Overexpression of 4-1BBL on a non-stimulatory cell line recapitulated the capacity to stimulate NK cells. However, subsequent experiments utilizing recombinant proteins expressed on beads suggested that 4-1BBL and IL-2 signals alone were insufficient to initiate robust NK cell proliferation. Other TNFSF members were also found to be involved and are being further explored.Conclusions Our study provides the first insights into the mechanism by which EBV-transformed B cells drive NK cell proliferation, highlighting the pivotal role of 4-1BB as a critical driver in this process. These findings offer valuable insights into the fundamental biology of NK cells, which not only inform the development of feeder cell-free clinical protocols for ex vivo NK cell expansion but also guide the design of innovative strategies to stimulate NK cell proliferation in vivo. Taken altogether, these insights hold the potential to lead to more effective and scalable NK cell-based treatment options for cancer patients.