Cytotoxic lymphocytes, including T cells and NK cells, are being developed as allogeneic, “off-the-shelf”, cell therapies for the treatment of hematological and solid tumors. Allogenic lymphocyte therapies face challenges, however, including limited expansion potential and limited in vivo persistence due to host immune rejection. To address these challenges, we developed an mRNA-reprogrammed iPSC line with a biallelic knockout of the beta-2 microglobulin (B2M) gene, a key component of MHC class I molecules, using an mRNA-encoded chromatin context-sensitive gene-editing endonuclease. We differentiated these B2M-knockout iPSCs using a novel, fully suspension process that replaces specialized micropatterned culture vessels with a spheroid culture step. The resulting lymphocytes were characterized for surface markers via flow cytometry and incubated with cancer cells to assess tumor cell engagement and cytotoxicity. Notably, we observed consistently higher yields of lymphocytes from the B2M-knockout iPSC line than from the parental wild-type iPSC line. Both wild-type and B2M-knockout lymphocytes cells killed 75-90% of K562 cells after 24 hours (effector to target (E:T) ratio of 5:1). Interestingly, cytotoxic lymphocytes derived from B2M-knockout iPSCs exhibited greater K562 cell killing with the addition of IL15 and IL2, while killing by wild-type cells was not controlled by these activating cytokines. Cancer cell killing activity was maintained through cryopreservation, albeit at a reduced level (15-40% reduction in activity). These results suggest that B2M-knockout iPSCs may serve as an ideal source of cytotoxic lymphocytes for the development of “off-the-shelf” allogeneic cell therapies for the treatment of cancer.