Abstract The Slam/SAP (SLAM-associated protein) signaling pathway plays a central role in the host immune response to infections. Males that inherit an inactivated SAP gene develop X-linked lymphoproliferative syndrome (XLP), a serious immunodeficiency that often results in enhanced susceptibility to a variety of pathogens. SAP is a cytosolic adapter protein that transduces signals from Slam family receptors, a family of nine (SLAMf1 to SLAMf9) cell surface receptors that are expressed only on hematopoietic cells. Although the Slam/SAP axis is known to regulate the development and function of several lymphocyte subsets, their role in γδ T cell biology is less well understood. We analyzed simultaneously the expression of multiple Slam receptors on γδ T cells and observed significant heterogeneity among γδ T cell subsets. Interestingly, Slam expression patterns marked functional γδ T cell subsets in both naïve and influenza-infected mice. SLAMf6 expression on lung γδ T cells correlated with IFNγ-producing CD27+ cells while SLAMf1 expression characterized IL-17-producing CD27− RORγt+ γδ T cells. The heterogeneous Slam receptor expression patterns of γδ T cell subsets can already be seen during embryonic thymic development, suggesting a possible role in the developmental programming of γδ T cell effector function. Disruption of Slam signaling through deletion of SAP affected the development of RORγt+ CD44hi Vγ4 T cells in the embryonic thymus, and the frequency of RORγt+ Vγ4 T cells in the periphery was still diminished in the adult. These results suggest that the Slam/SAP signaling pathway is an important regulator of the functional programming γδ T cells undergo during thymic development which affects mucosal immune responses later in life.