Granulocyte colony-stimulating factor (G-CSF) is the cytokine critical for directing neutrophilic granulocyte differentiation. Early G-CSF signaling events in myeloid cells involves activation of STATs, proteins that serve the dual function of signal transduction and activation of transcription, especially the activation of Stat3. A dominant-negative mutant construct of Stat3 inhibited G-CSF-mediated neutrophilic differentiation indicating that Stat3 is a essential component for driving the G-CSF-mediated differentiation program in myeloid cells. Three isoforms of Stat3 have been identified, alpha(p92), beta(p83) and gamma(p72) each derived from a single gene. Stat3alpha is the predominant isoform expressed in most cells. Stat3beta is derived from Stat3alpha by alternative RNA splicing. Stat3gamma is derived from Stat3alpha by limited proteolysis. Mapping of Stat3alpha and Stat3beta activation in M1 murine myeloid leukemia cells revealed that their optimal activation required G-CSFR constructs containing both Y704 and Y744. These amino acid residues has previously been demonstrated to be essential for G-CSF-induced differentiation in this cells. Phosphopeptide affinity and phosphopeptide inhibition studies indicate that Stat3alpha and Stat3beta are recruited to the G-CSF receptor complex through their interaction with the receptor at phosphotyrosines Y704 and Y744. Y744 is followed at the +3 position by Cys (C). This sequence YXXC, represents a novel motif implicated in the recruitment and activation of Stat3alpha, Stat3beta and Stat3gamma by the hG-CSFR. Structurally, Stat3alpha, Stat3beta and Stat3gamma differ from each other in their C-terminal transactivation domain. In the beta isoform, the Stat3alpha transactivation domain is replaced by 7 amino acid residues which enable Stat3beta to interact with c-Jun. In the gamma isoform, the Stat3alpha transactivation domain is removed by limited proteolysis creating a dominant negative isoform. In immature human myeloid cells capable of differentiating into neutrophils in response to G-CSF, G-CSF did not activate Stat3alpha; rather. it activated predominantly Stat3beta. These findings combined with recent reports linking Stat3alpha with proliferation and transformation suggest that the beta isoform of Stat3 may be more critical for G-CSF-mediated differentiation. Activation of Stat3gamma occurred predominantly in terminally differentiated neutrophils suggesting that it may be part of a controlled proteolytic mechanism modulating pro-proliferative protein(s) in mature myeloid cells.