Mutations in either syntaxin 11 (Stx11) or Munc18-2 abolish cytotoxic T lymphocytes (CTL) and natural killer cell (NK) cytotoxicity, and give rise to familial hemophagocytic lymphohistiocytosis (FHL4 or FHL5, respectively). Although Munc18-2 is known to interact with Stx11, little is known about the molecular mechanisms governing the specificity of this interaction or how in vitro IL-2 activation leads to compensation of CTL and NK cytotoxicity. To understand how mutations in Munc18-2 give rise to disease, we have solved the structure of human Munc18-2 at 2.6 Å resolution and mapped 18 point mutations. The four surface mutations identified (R39P, L130S, E132A, P334L) map exclusively to the predicted syntaxin and soluble N-ethylmaleimide–sensitive factor accessory protein receptor binding sites of Munc18-2. We find that Munc18-2 binds the N-terminal peptide of Stx11 with a ∼20-fold higher affinity than Stx3, suggesting a potential role in selective binding. Upon IL-2 activation, levels of Stx3 are increased, favoring Munc18-2 binding when Stx11 is absent. Similarly, Munc18-1, expressed in IL-2–activated CTL, is capable of binding Stx11. These findings provide potential explanations for restoration of Munc18-Stx function and cytotoxicity in IL-2–activated cells.