Thymocytes are selected to mature according to their ability to interact with self major histocompatibility complex (MHC)–peptide complexes displayed on the thymic stroma. Using two-photon microscopy, we performed real-time analysis of the cellular contacts made by developing thymocytes undergoing positive selection in a three-dimensional thymic organ culture. A large fraction of thymocytes within these cultures were highly motile. MHC recognition was found to increase the duration of thymocyte–stromal cell interactions and occurred as both long-lived cellular associations displaying stable cell-cell contacts and as shorter, highly dynamic contacts. Our results identify the diversity and dynamics of thymocyte interactions during positive selection.