It has been unclear whether the progenitors colonizing the thymus are multipotent or T cell lineage restricted. We investigated the developmental potential of hematopoietic progenitors in various populations of liver and blood cells from day 12 fetuses using the recently established in vitro experimental system effective in determining the capability of individual progenitors to generate T, B, and myeloid cells. Multipotent progenitors (p-Multi) were exclusively found in the Sca-1 high-positive (Sca-1high) subpopulation of lineage marker (Lin)-c-kit+CD45+ fetal liver cells. Restriction of developmental capacity begins at the Sca-1high stage, and a large majority of progenitors in the Sca-1low or Sca-1- population are restricted to generate T, B, or myeloid cells. Such a lineage commitment or restriction taking place in the fetal liver is independent of the thymus, because no difference in the proportion of different types of progenitors were seen between nu/nu and nu/+ fetuses. T cell lineage-restricted progenitors (p-T) were abundant in the blood of day 12 fetuses, whereas p-Multi were undetectable. It was further shown that the p-Multi generated a large number of B and myeloid cells in the thymic lobe. These results strongly suggest that it is p-T but not p-Multi that migrate into the thymus.