Neonatal CD4 naive T cells require combined IL-10 and IL-1β signaling in addition to TGF-β and IL-4 to differentiate into IL-9–producing cells. Naive or memory CD4 T cells were purified from cord blood or from the peripheral blood of adult subjects and stimulated under Th9, Th1, Th2, and Th17 cell-inducing conditions or in the presence of different cytokine combinations as indicated for 5 days. (A) Cytoplasmic cytokines were analyzed by intracellular flow cytometry after restimulation of the cells with phorbol myristate acetate and ionomycin. Results from 1 of 6 independent experiments with cells from different donors are shown. (B) Cytokine mRNA was assessed by real-time PCR; mRNA levels were normalized for the expression of cyclophilin. Mean ± SEM of 5 independent experiments with cells from different donors is shown. (C) Frequencies of IL-9–producing cells were assessed by intracellular flow cytometry. The mean ± SEM of 4 independent experiments with cells from different donors is demonstrated. **P < .01 by Student t test compared with medium control without any cytokine.