In a manner similar to many other cytokines, treatment of cells with granulocyte CSF (G-CSF) has been shown to induce the tyrosine phosphorylation of the STAT proteins. Activation of Stat1 and Stat5 by G-CSF requires the membrane-proximal cytoplasmic domain of the receptor, including box1 and box2, while G-CSF-stimulated tyrosine phosphorylation of Stat3 also requires a region distal to box 2. In this study, we show that although the membrane-proximal 55 amino acids of the G-CSF receptor are sufficient for activation of Stat5, the maximal rate of Stat5 activation requires an additional 30 amino acids of the cytoplasmic domain. In contrast, the distal carboxyl-terminal region of the receptor appears to down-regulate Stat5 activation in that deletion of this carboxyl terminus results in increased amplitude and prolonged duration of Stat5 activation by G-CSF. Significantly, expression of a truncated dominant-negative Stat5 protein in hemopoietic cells not only inhibits G-CSF-dependent cell proliferation, but also suppresses cell survival upon G-CSF withdrawal. We further show that a potential protein tyrosine phosphatase may play a critical role in the down-regulation of G-CSF-stimulated Stat5 activation. These results demonstrate that two distinct cytoplasmic regions of the G-CSF receptor are involved in the regulation of the intensity and duration of Stat5 activation, and that Stat5 may be an important player in G-CSF-mediated cell proliferation and survival.