Interleukin-4 (IL-4) is the main cytokine that polarizes activated naïve CD4+ T cells in the T helper 2 (Th2) direction. IL-4 also regulates the subsequent stages of Th2 cell mediated diseases, such as allergies. We have conducted a proteomics study to identify IL-4 induced differences during the initial stages of T helper cell differentiation. Primary CD4+ T lymphocytes were isolated from human cord blood, activated through CD3 and CD28, and cultured in the presence or absence of IL-4. Soluble proteins were separated by two-dimensional electrophoresis and visualized by staining with autoradiography, which indicated that at least twenty proteins might be regulated by IL-4. From this minimum of twenty stained proteins, altogether thirty-five proteins were identified using tandem mass-spectrometry. Interestingly, the fragmented form of Ly-GDI, a known target of Caspase-3, was observed to be downregulated in IL-4 treated cells. It was shown in further studies that IL-4 decreases Caspase-3 activity and cell death in these cells. Neutralizing Fas-FasL interaction led to decreased Caspase-3 activity and lowered Ly-GDI fragmentation. We further characterized the effects of IL-4 on the expression of main regulators in the Fas-mediated pathway. We demonstrate that IL-4 decreases expression of Fas receptor and increases expression of Bid, Bcl-2, and Bcl-xL. Importantly, IL-4 significantly upregulates the short form of c-FLIP, although the levels of c-FLIP long are unaltered after IL-4 induction. Taken together, our results indicate that IL-4 inhibits caspase activity during the initial stages of human Th2 cell differentiation by regulating expression of several key players in the Fas-induced pathway.