Unconventional or innate-like T cells include γδ T, natural killer T (NKT) and mucosal-associated invariant T (MAIT) cells. MAIT and γδ T cells are a major component of the human immune system, comprising 1-15% of peripheral blood lymphocytes and have been implicated in responses to many pathogens. However, our knowledge of their precise role in protective immunity and the molecular mechanisms regulating their development, activation and function is limited. Human primary immunodeficiencies caused by loss-of-function mutations in single genes provide a unique opportunity to assess the requirement for particular molecules as regulators of human unconventional T-cell activation and differentiation. Autosomal dominant-hyper IgE syndrome (AD-HIES) is caused by heterozygous mutations in STAT3. We used lymphocytes from these patients to assess the roles of STAT3 in NKT, MAIT and γδ T cell differentiation in vivo and in vitro. Patients with STAT3 mutations have normal numbers of γδ T cells but reduced frequencies of MAIT and NKT cells ex vivo. This resulted from a T-cell intrinsic requirement for STAT3 function in regulating these lymphocyte subsets. Further, STAT3-deficient γδ T cells and MAIT cells displayed altered responses to in vitro stimulation, with impaired production of Th17-type effector cytokines. Thus, STAT3 plays an important role in regulating the numbers and the function of unconventional T cells. Defects in these cells may contribute to the clinical infectious phenotype that is characteristic of AD-HIES patients.