T-cell motility is essential for the T cells’ ability to scan antigens within lymph nodes and initiate contact with antigen-presenting cells. While T-cell migration has been extensively studied using in vitro migration assays, accumulating evidence indicates that the T-cell migration within lymph nodes is modulated by the surrounding cells and extracellular matrix, which form the confined architecture of the lymph nodes. Therefore, to understand the mechanisms of T-cell motility in vivo, their cell migration must be analyzed under physiological conditions. To this end, two-photon microscopy is extremely useful; this technique enables the tracking of fluorescently labeled cells in vivo and ex vivo, with high spatial and temporal resolutions. Here we describe the experimental procedures for applying two-photon microscopy to the in vivo and ex vivo imaging of T-cell migration in mouse lymph nodes. These approaches provide physiological insight into the mechanisms of T-cell behavior at a single-cell level in the three-dimensional lymph node environment.