ETP were isolated from E14.5 fetal thymi and seeded at a density of 3000 cells per well onto OP9-DL1 cells (plated the previous day at a density of 2 × 104 per well) or iTEC (density 2 × 105) in 24 well plates. The OP9-DL1/ETP co-cultures were subsequently cultured in medium containing IL7 (5 ng ml−1) and Flt3L (5 ng ml−1) (Porrit et al., 2004) until day 6 of culture, then IL7 (1 ng ml−1) and Flt3L (5 ng ml−1) until the end of the experiment, while the iTEC/ETP and control MEF/ETP co-cultures were cultured in medium containing IL7 (1 ng ml−1) and Flt3L (5 ng ml−1) throughout. Cultures were analyzed by flow cytometry with the markers shown after 4, 8 or 12 days of co-culture. n = 1. Note that the iTEC used for the above experiments were made by isolating MEFs from E13.5 Rosa26CAG−STOP−Foxn1−IRES−GFP embryos; these MEFs were expanded in culture for 4 days, then treated with 1 μM 4OHT for 48 hours. The following day, GFP+ cells were isolated by flow cytometric cell sorting, then plated into 24 well plates at a density of 2 × 105 cells per well. They were seeded with ETPs 3 days later. This protocol differs slightly from the protocol used in Fig. 2.