Granulocyte-colony stimulating factor (G-CSF) induces proliferation of myeloid progenitor cells and controls their differentiation into mature neutrophils. Signal transducer and activator of transcription (STAT) proteins STAT3 and STAT5 are activated by G-CSF and play distinct roles in neutrophil development. Suppressor of cytokine signaling (SOCS) proteins are induced by STATs and inhibit signaling through various negative-feedback mechanisms. SOCS proteins can compete with docking of signaling substrates to receptors, interfere with Janus tyrosine kinase activity, and target proteins for proteasomal degradation. The latter process is mediated through the conserved C-terminal SOCS box. We determined the role of various SOCS proteins in controlling G-CSF responses and investigated the involvement of the SOCS box therein. We show that SOCS1 and SOCS3, but not CIS and SOCS2, inhibited G-CSF-induced STAT activation in human embryo kidney 293 cells. In myeloid 32D cells, SOCS1 and SOCS3 are induced by G-CSF. However, relative to interleukin-3-containing cultures, during G-CSF-induced neutrophilic differentiation, SOCS3 expression was further elevated, while SOCS1 levels remained constant. SOCS box deletion mutants of SOCS1 and SOCS3 were severely hampered in their abilities to inhibit STAT activation and to efficiently suppress colony formation by primary myeloid progenitors in response to G-CSF. These data demonstrate the importance of the SOCS box for the inhibitory effects of SOCS proteins on G-CSF signaling and show that among the different SOCS family members, SOCS3 is the major negative regulator of G-CSF responses during neutrophilic differentiation.