NK cells recognize cancer and viral cells by binding their activating receptors to antigens presenting on the membrane of target cells. Although the activation mechanism of NK cells is a subject of extensive research today, the role of the composition and spatial distribution of activating ligands in NK cell cytotoxicity is barely understood. In this work, we engineered a nanochip whose surface was patterned with matrices of antigens for NKG2D activating receptors. These matrices mimicked the spatial order of the surface of antigen presenting cells with molecular resolution. Using this chip, we elucidated the effect of the antigen spatial distribution on the NK cell spreading and immune activation. We found that the spatial distribution of the ligand within the 100 nm length-scale provides the minimal conditions for NKG2D regulated cell spreading. Furthermore, we found that the immune activation of NK cells requires the same minimal spatial distribution of activating ligands. Above this threshold, both spreading and activation plateaued, confirming that these two cell functions work hand in hand. Our study provides an important insight on the spatial mechanism of the cytotoxic activity of NK cells. This insight opens the way to rationally designed antitumor therapies that harness NK cytotoxicity.