Failure of Pten-deficient immature Vα14iNKT cells to mature in vitro. CD44^highNK1.1⁻ (stage 2, immature) Vα14iNKT cells were purified from the thymi of Pten^flox/flox or LckCrePten^flox/flox mice and injected into FTOCs derived from a Jα18^−/− embryo. (Left) Flow cytometric analysis of cell populations injected. (Right) At 9 days after injection, Vα14iNKT cells in the FTOC were isolated and analyzed by flow cytometry to detect stage 3 (mature) cells. Mature Vα14iNKT cells were very rare in FTOCs receiving Pten-deficient stage 2 NKT cells. One result representative of 3 experiments is shown.