Natural killer (NK) cell function is regulated by inhibitory receptors, such as the family of killer immunoglobulin-like receptors (KIRs) and the NKG2A/CD94 heterodimer. These receptors recognize cognate HLA class I molecules on potential target cells, and recent studies imply that an HLA-B dimorphism at position -21 in the gene segment encoding the leader peptide dictates whether NK cell regulation primarily relies on the KIRs or the NKG2A/CD94 receptor. The impact of this HLA-B dimorphism on NK cell-mediated destruction of leukemic cells or on the course of leukemia is largely unknown. In a first part of this study, we compared functions of NK cells in subjects carrying HLA-B -21M or 21T using interleukin-2 (IL-2)-activated NK cells and leukemic cells from patients with acute myeloid leukemia (AML). Subjects carrying HLA-B -21M harbored better-educated NKG2A+ NK cells and displayed superior capacity to degranulate lytic granules against KIR ligand-matched primary leukemic blasts. Second, we aimed to define the potential impact of HLA-B -21 variation on the course of AML in a phase 4 trial in which patients received IL-2-based immunotherapy. In keeping with the hypothesis that 21M may be associated with improved NK cell functionality, we observed superior leukemia-free survival and overall survival in -21M patients than in -21T patients during IL-2-based immunotherapy. We propose that genetic variation at HLA-B -21 may determine the antileukemic efficacy of activated NK cells and the clinical benefit of NK cell-activating immunotherapy.