In vivo imaging of thymocytes has not been accomplished due to their localization deep within opaque body and high susceptibility to surgical stress. To overcome these problems, medaka is useful because of transparency and ex-uterine development. We report the noninvasive detection of thymocytes in transgenic medaka that express fluorescent protein under the control of immature-lymphocyte-specific rag1. We show that lymphoid progenitor cells colonize the thymus primordium in an anterior-to-posterior orientation-specific manner, revealing that extrathymic anterior components guide prevascular thymus colonization. We also show that developing thymocytes acquire "random walk motility" along with the expression of Ag receptors and coreceptors, suggesting that thymocyte walking is initiated at the developmental stage for repertoire selection. Thus, transgenic medaka enables real-time intravital imaging of thymocytes without surgical invasion.