NK cells are critical for the recognition and lysis of herpesvirus-infected cells. Patients with NK cell immunodeficiency may suffer from unusually severe and/or recurrent herpesvirus infections; however, the genetic cause is frequently unknown. PLCG2 encodes a signaling protein in NK cell and B cell receptor signaling, in which dominant-negative or gain-of-function mutations may cause cold urticaria, antibody deficiency, or autoinflammation. However, loss-of-function mutations and PLCG2 haploinsufficiency have never been reported in human disease. We examined 2 families with autosomal dominant NK cell immunodeficiency with dual high-dimensional techniques, mass cytometry and whole-exome sequencing, to identify the cause of disease. We identified two novel heterozygous loss-of-function mutations in PLCG2 that impaired NK cell function, including calcium flux, granule movement, and target killing. Although expression of mutant PLCG2 protein in vitro was normal, phosphorylation of both mutants was diminished. In contrast to PLAID and APLAID, B cell function remained intact. Plcg2+/- mice, as well as targeted CRISPR knock-in mice, also displayed impaired NK cell function with preserved B cell function, phenocopying human PLCG2 haploinsufficiency. We report the first known cases of PLCG2 haploinsufficiency, a clinically and mechanistically distinct syndrome from previously reported mutations. Therefore, these families represent a novel disease, highlighting a role for PLCG2 haploinsufficiency in herpesvirus-susceptible patients and expanding the spectrum of PLCG2-related disease.