Exposure to irradiated EBV-transformed B lymphoblastoid cell lines (LCL) in the presence of IL-2 triggers extensive proliferation of human NK cells. Previously, we used this knowledge to design a GMP protocol to expand NK cell numbers for infusion into cancer patients in a clinical trial. Remarkably, the molecular interactions which trigger this proliferation remained unknown. 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. Results were confirmed by CRISPR Cas9-mediated gene editing of LCL to disrupt NK cell ligands and subsequent co-culture assays of NK cell proliferation. These experiments identified a primary role of TNFSF9, which is augmented on LCL by irradiation, interacting with TNFRSF9 on NK cells to trigger entry into cell cycle. Overexpression of TNFSF9 on mouse P815 cell line recapitulated the capacity to stimulate NK cells. However, subsequent experiments utilizing recombinant proteins expressed on beads suggested that TNFSF9 and IL-2 signals alone were insufficient to initiate robust NK cell proliferation. In addition to providing basic knowledge about NK cells, this study informs future feeder cell-free clinical protocols to expand NK cells and the design of novel interventions to induce NK cell proliferation in vivo.