Composition and phenotype of CD45^–thymic stromal cells changes with age. At various time points, CD45^– stromal cells were stained for (A) MHC II versus EpCAM, (B) MHC II versus MTS-15, and (C) MHC II versus Ly51. Regions indicate CD45^– stroma composed of TECs (MHC II^hi and MHC II^lo at adult time points), fibroblasts, cTECs (MHC II⁺/Ly51⁺), and mTECs (MHC II⁺/Ly51^–). All dot plots are gated on CD45^– thymic stromal cells isolated from at least 3 thymi pooled at the ages indicated and are representative of 2 to 5 experiments. Bar graphs of the ratios of (D) TEC/fibroblasts, (E) mTEC/cTEC, and (F) MHC II^hi/MHC II^lo TECs in 4-week-old and middle-aged (12-month-old) thymi. Means and standard errors are plotted, and groups were compared using a Mann-Whitney rank sum U test. (*P < .05, **P < .01, ***P < .001). (G) Cytospins of purified cTECs, mTEC^hi subset, and mTEC^lo subset from 4- to 6-week-old mice stained with anti-K5 (green) and anti-K8 (red). (H) The mean proportion of TEC subsets that expressed K5, K8, or both (double positive, DP) with standard error from 3 experiments.