Natural killer (NK) cells provide a unique barrier to semiallogeneic bone marrow (BM) transplantation. In the setting where the parents donate to the F1 offspring, rejection of parental bone marrow occurs. This "hybrid resistance" is completely NK cell dependent, as T cells in the F1 recipient tolerate parental grafts. Previously, we demonstrated that rejection of BALB/c parental BM by (BALB/c × C57BL/6) F1-recipient NK cells is dependent on the NKG2D-activating receptor, whereas rejection of parental C57BL/6 BM does not require NKG2D. BALB/c and B6 mice possess different NKG2D ligand genes and express these ligands differently on reconstituting BM cells. Herein, we show that the requirement for NKG2D in rejection depends on the major histocompatibility complex haplotype of donor cells and not the differences in the expression of NKG2D ligands. NKG2D stimulation of NK cell-mediated rejection was required to overcome inhibition induced by H-2D(d) when it engaged an inhibitory Ly49 receptor, whereas rejection of parental BM expressing the ligand, H-2K(b), did not require NKG2D. Thus, interactions between the inhibitory receptors on F1 NK cells and parental major histocompatibility complex class I ligands determine whether activation via NKG2D is required to achieve the threshold for rejection of parental BM grafts.