Neural crest (NC) cells are multipotent cells that can differentiate into melanocytes, neurons, glias and myofibroblasts. They migrate into the fetal thymus on embryonic day (E) 12 in mice and may participate in thymic organogenesis. Although the abnormality of migration and distribution of NC cells in the thymus results in immunodeficiency, the spatial and temporal presence of their progeny cells has not been defined in detail. In this study, we traced NC-derived cells based on the myelin protein zero gene promoter-Cre-mediated excision. We demonstrated that large numbers of NC-derived cells in the thymus were detected on E11.5 to E16.5 but rarely on E17.5. A colony formation assay of single thymic cells demonstrated that multipotent cells with the potential to differentiate into melanocytes, neurons and/or glias were present in the E14.5 and E15.5 but not in the E17.5 fetal thymus. Furthermore, we confirmed that these multipotent cells were NC-derived cells. Taken together, these findings imply that multipotent NC-derived cells are present in the developing thymus, but rarely in this organ at a later stage, suggesting that NC-derived cells may play roles in thymic organogenesis at an early embryonic stage.