Functional characteristics of neonatal RTEs are not solely the result of the developmental age of the hematopoietic stem cell. Day 14 FL cells or adult BM cells from RAG2p-GFP^+/− mice were transplanted intravenously into lethally irradiated wild-type RAG2p-GFP^−/− adults. Six to 7 weeks later, LN CD4⁺ cells were purified and then sorted to obtain GFP^hi RTEs. (A) Thymic reconstitution was similar in mice who received either FL (left panel) or BM cells (right panel). Both groups also possessed similar levels of GFP^hi RTEs in the peripheral LN CD4⁺ compartment. Purity of sorted RTEs was > 99%. (B) Sorted RTEs were activated with PBαCD3 and αCD28 for 48 hours. Supernatants were then harvested for cytokine-specific ELISA. Data represent pooled data from 3 independent experiments and are shown as the mean ± SEM; n = 5 or 6 mice per group (FL and BM recipients). (C) Sorted RTEs were cultured in complete media or stimulated with either 10 ng/mL IL-7 or PBαCD3 and αCD28 for 48 hours. ³H-thymidine was added for the last 24 hours of culture to measure proliferation. Graphs represent pooled data from 3 independent experiments; n = 5 or 6 mice per group. All data are shown as the mean ± SEM.