Two-photon microscopy makes it possible to image in real-time fluorescently labeled cells located in deep tissue environments. We describe a procedure to visualize the behavior of lymph node T cells during either priming or tolerance, in live, anesthetized mice. Intravital imaging of T lymphocytes is a powerful tool to study the cellular orchestration of adaptive immune responses in physiological settings. This method should provide new insights into the regulation of lymphocyte migration and cell-cell interactions in various immunological contexts.