After adoptive transfer, in vitro–cultured Treg cells survive, traffic to secondary lymphoid organs, and preferentially expand at sites of antigenic challenge in vivo. Unlabeled naive CD4⁺CD25⁻ T cells (1 × 10⁶) were coinjected in T cell–depleted CBA mice with 2 × 10⁶ CD25⁺ line labeled with 5 μM CFSE (A) or with 2 × 10⁶ GFP-CD25⁺ line (B). The day after adoptive transfer, recipients received skin grafts from CBK donors. Survival, trafficking, and divisions of the transferred Treg cells were assessed for individual animals by flow cytometric analysis of separate cell suspensions from spleen, graft-draining LNs, and distant mesenteric LNs at various time points after transplantation—days 7, 14, and 21 for CFSE analysis (here shown at day 14) and days 20 and 60 for GFP analysis (here shown at day 60). For the animals that underwent cotransfer with the GFP-CD25⁺ line, cell suspensions were also obtained from the grafted tissue and distant nongrafted skin for flow cytometric analysis. Histograms correspond to a minimum of 30 000 acquired events in a mixed lymphocyte gate consisting of live CD4⁺ T cells. Data are shown from 1 of 5 representative mice per group.