Mouse natural killer (NK) cells acquire effector function by an education process termed “licensing” mediated by inhibitory Ly49 receptors which recognize self-MHC class I. Ly49 receptors can bind to MHC class I on targets (in trans) and also to MHC class I on the NK-cell surface (in cis). Which of these interactions regulates NK-cell licensing is not yet clear. Moreover, there are no clear phenotypic differences between licensed and unlicensed NK cells, perhaps because of the previously limited ability to study NK cells with synchronized licensing. Here, we produced MHC class I-deficient mice with inducible MHC class I consisting of a single-chain trimer (SCT), ovalbumin peptide-β2 microgloblin-H2K^b (SCT-K^b). Only NK cells with a Ly49 receptor with specificity for SCT-K^b were licensed after MHC class I induction. NK cells were localized consistently in red pulp of the spleen during induced NK-cell licensing, and there were no differences in maturation or activation markers on recently licensed NK cells. Although MHC class I-deficient NK cells were licensed in hosts following SCT-K^b induction, NK cells were not licensed after induced SCT-K^b expression on NK cells themselves in MHC class I-deficient hosts. Furthermore, hematopoietic cells with induced SCT-K^b licensed NK cells more efficiently than stromal cells. These data indicate that trans interaction with MHC class I on hematopoietic cells regulates NK-cell licensing, which is not associated with other obvious phenotypic changes.