Natural killer (NK) cells have been thought to develop from committed progenitors in the bone marrow. However, a novel pathway of thymus-dependent NK-cell development that produces a unique subset of NK cells expressing CD127 has recently been reported. We now have identified 2 populations of NK progenitors, one in the thymus and the other in the lymph node (LN). Immature double-negative 2 (CD4(-)CD8(-)CD44(+)CD25(+)) thymocytes have potential to produce NK cells with rearranged T-cell receptor gamma genes (Tcrgamma(+)) in vitro. Tcrgamma(+) NK cells are rare in spleen but relatively abundant in the thymus and LN. Approximately 20% of LN NK cells are Tcrgamma(+), and they are found at similar levels in both CD127(+) and CD127(-) subsets. Moreover, a subpopulation of LN cells resembling immature thymocytes differentiates into Tcrgamma(+) NK cells in vitro and also repopulates the NK compartment in lymphopenic mice. Athymic mice lack the LN NK progenitors expressing CD127 as well as Tcrgamma(+) NK cells. These results suggest that Tcrgamma(+) NK cells may be generated from unique progenitors in the thymus as well as in the LN.