The ability of antibodies to recruit Natural Killer (NK) cells to kill target cells is a cornerstone of therapeutics being developed for infectious diseases and cancer. The IgG receptor CD16a is responsible for activating NK cell antibody dependent cellular cytotoxicity (ADCC), but a detailed molecular mechanism of how this occurs is lacking, limiting the potential of therapeutic development. We sought to improve our understanding of the role of CD16a during ADCC using super-resolution MINFLUX nanoscopy and single particle cryo-electron microscopy EM. We activated NK-92 cells on supported lipid bilayers using Trastuzumab and localized endogenously labeled CD16a using MINFLUX with a localization precision of ~2.5 nm. We show that CD16a is predominantly present in pairs, potentially as homodimers, that are ~17-18 nm apart and do not significantly change from resting to activated cells. We found that a nanobody (C28nb) against CD16a blocks NK cell ADCC activity in vitro despite not competing for IgG binding. Using cryo-EM, we show that C28nb binds to the membrane distal domain 1 (D1) of CD16a, overlapping with the L48H/R epitope that is shown to improve NK cell ADCC function. Functional analysis suggests that the C28nb epitope mediates CD16a interaction with CD2, and we are exploring the mechanistic basis of how this regulates NK cell ADCC. We next plan to expand our hybrid cryo-EM/MINFLUX approach to explore the spatial relationship of CD16a to CD2 and other proteins implicated in ADCC across biological scales, which will shed light on observed disparities in antibody ADCC potency and heterogeneities in NK cell cytotoxicity.