The advancement of NK cell-based immunotherapy requires platform-specific approaches that overcome intrinsic functional limitations while enabling flexible adaptation across diverse manufacturing settings. NK101, a human NK cell line phenotypically aligned with the CD56^dimCD62L⁺ intermediate-stage subset, retains functional plasticity and robust cytokine responsiveness, providing a versatile platform for adoptive immunotherapy development. Although baseline cytotoxicity is relatively modest, this profile allows for rational reprogramming through targeted cytokine and genetic interventions. We initiated this reprogramming by screening IL-2 family cytokines for their effects on proliferation and effector molecule production. IL-2 and IL-15 promoted proliferation and IFN-γ secretion, whereas IL-21 uniquely enhanced granzyme B levels with minimal effects on proliferation and IFN-γ. Co-stimulation with IL-2 and IL-21 proved most effective, maximizing cytotoxic potential while sustaining proliferation and enhancing IFN-γ secretion. Transcriptomic profiling further highlighted IL-21’s role in activating granzyme-mediated apoptosis, which was amplified by IL-2 co-priming. These findings were applied to genetically engineered NK101 cells expressing high-affinity CD16 (NK101-16V), where IL-2 and IL-21 co-priming enhanced both baseline cytotoxicity and antibody-dependent cellular cytotoxicity. Additional engineering to express membrane-bound TRAIL (NK101-16V-TR) significantly augmented TRAIL-mediated apoptosis, enabling greater killing of resistant tumors. In vivo, IL-2/IL-21-primed NK101-16V-TR cells in combination with rituximab achieved complete tumor regression in all Jeko-1 xenograft-bearing mice. Collectively, this study presents a broadly applicable design framework for NK cell manipulation, integrating transient cytokine priming with multi-functional genetic modification to guide the development of next-generation NK cell therapies.