The immune system exerts a critical function as it recognizes and eliminates transformed or neoplastic cells, a process also referred to as immunosurveillance. NK cells play a particularly important role in that they are able to recognize tumor cells via "missing-self"-i.e., the absence of major histocompatibility complex Class I on target cells. Moreover, recent studies suggest that NK cells also participate in the onset and regulation of adaptive immune responses. The exact molecular pathways by which this occurs, however, remain poorly understood. To obtain further insight into the genes that are required for self-induced immune responses via NK cell-mediated cell death, our laboratory initiated a forward genetic approach using N-ethyl-N-nitrosourea (ENU) as a mutagen. Specifically, we tested the ability of NK cells from G3 ENU germline mice to recognize missing-self target cells and induce CD8+ T-cell responses following immunization with irradiated tumor cells. Here we present two ENU germline mutants, designated Ace and Chip, that are defective in the recognition of β-2 microglobulin-deficient target cells, yet exhibit improved clearance of B16 melanoma cells in vivo. Coarse mapping and whole genome sequencing of the Chip mutation revealed a missense mutation causing a T'A amino acid substitution in the highly conserved third immuno-receptor tyrosine-based switch motif of CD244 (2B4). The forward genetic approach described here promises to reveal important insight into critical genes that are required for host responses involved in anticancer immunity.