Thymocytes must complete an elaborate developmental program in the thymus to ultimately generate T cells that express functional but neither harmful nor useless TCRs. Each developmental step coincides with dynamic relocation of the thymocytes between anatomically discrete thymic microenvironments, suggesting that thymocytes' migration is tightly regulated by their developmental status. Chemokines produced by thymic stromal cells and chemokine receptors on the thymocytes play an indispensable role in guiding developing thymocytes into the different microenvironments. In addition to long-range migration, chemokines increase the thymocytes' motility, enhancing their interaction with stromal cells. During the past several years, much progress has been made to determine the various signals that guide thymocytes on their journey within the thymus. In this review, we summarize the progress in identifying chemokines and other chemoattractant signals that direct intrathymic migration. Furthermore, we discuss the recent advances of two-photon microscopy in determining dynamic motility and interaction behavior of thymocytes within distinct compartments to provide a better understanding of the relationship between thymocyte motility and development.