Natural killer (NK) cells are a critical first line of defense against viral infection. Rare mutations in a small subset of transcription factors can result in decreased NK cell numbers and function in humans, with an associated increased susceptibility to viral infection. However, our understanding of the specific transcription factors governing mature human NK cell function is limited. Here we use a non-viral CRISPR-Cas9 knockout screen targeting genes encoding 31 transcription factors differentially expressed during human NK cell development. We identify myocyte enhancer factor 2C (MEF2C) as a master regulator of human NK cell functionality ex vivo. MEF2C-haploinsufficient patients and mice displayed defects in NK cell development and effector function, with an increased susceptibility to viral infection. Mechanistically, MEF2C was required for an interleukin (IL)-2- and IL-15-mediated increase in lipid content through regulation of sterol regulatory element-binding protein (SREBP) pathways. Supplementation with oleic acid restored MEF2C-deficient and MEF2C-haploinsufficient patient NK cell cytotoxic function. Therefore, MEF2C is a critical orchestrator of NK cell antiviral immunity by regulating SREBP-mediated lipid metabolism.