Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that recognizes glycolipid antigens presented by the major histocompatibility complex class I–related protein CD1d. Although iNKT cells have received a lot of attention as targets for the development of immunotherapies, few studies have investigated the in vivo response of iNKT cells to glycolipid antigen activation. Accumulating evidence indicates that iNKT cells generate a dynamic response to in vivo activation by glycolipid antigens that is characterized by surface receptor downmodulation, expansion, cytokine production, cross talk with other cells, homeostatic contraction, and acquisition of an anergic phenotype. These studies provide new insight into the biology of iNKT cells and have important implications for designing safe and effective iNKT cell–based vaccines and therapies.