IL-3 plays important roles in the growth and survival of hematopoietic progenitor cells, processes modeled in studies of the IL-3-dependent cell line Ba/F3. To gain insights into molecular mechanisms governing cell fate, we examined the patterns of proteins up-regulated following stimulation of Ba/F3 cells with IL-3. Through two-dimensional electrophoresis and proteomics-based approaches, we identified 11 proteins. Of these, expression of 14-3-3gamma was significantly increased by IL-3 stimulation at both the transcriptional and translational levels. 14-3-3gamma overexpression in Ba/F3 cells abrogated dependence on IL-3 and was associated with activation of PI3K and MAPK signaling cascades, suggesting that the functions of 14-3-3gamma in normal hematopoietic progenitors are to promote survival and growth through the activation of distinct signaling pathways. Additionally, the up-regulation of Bax and Bad was seen with the ablation of 14-3-3gamma, resulting in cell death. These results indicate that deregulated expression of 14-3-3gamma may contribute to malignant transformation, possibly providing a new target for therapeutic intervention in hematopoietic neoplasms.