Although many aspects of CD4⁺CD25⁺ T regulatory (T_reg) cell development remain largely unknown, signaling through the IL-2R represents one feature for the production of T_reg cells. Therefore, the present study was undertaken to further define early developmental steps in the production of T_reg cells, including a more precise view on the role of interleukin (IL)-2 in this process. After adoptive transfer of wild-type T_reg cells into neonatal IL-2Rβ^−/− mice, only a small fraction of donor T_reg cells selectively seeded the lymph node (LN). These donor T_reg cells underwent rapid and extensive IL-2–dependent proliferation, followed by subsequent trafficking to the spleen. Thus, IL-2 is essential for T_reg cell proliferation in neonatal LN. The number and distribution of T_reg cells in the periphery of normal neonatal mice closely paralleled that seen for IL-2Rβ^−/− mice that received T_reg cells. However, for normal neonates, blockade of IL-2 decreased T_reg cells in both the thymus and LN. Therefore, two steps of T_reg cell development depend upon IL-2 in neonatal mice, thymus production, and subsequent expansion in the LN.