CD1d-restricted natural killer T (NKT) cells are important for host defense against a variety of microbial pathogens. How and when these T cells become activated physiologically during infection remains unknown. Our data support a model in which NKT cells use a unique activation mechanism not requiring their recognition of microbial antigens. Instead, weak responses to CD1d-presented self antigens were amplified by interleukin 12 made by dendritic cells in response to microbial products, resulting in potent interferon-gamma secretion. NKT cells were among the first lymphocytes to respond during Salmonella typhimurium infection, and their activation in vivo also depended on interleukin 12 and CD1d recognition. We propose this mechanism of activation as a major pathway responsible for the rapid activation of NKT cells in different microbial infections.