Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) stimulate the proliferation and maturation of myeloid progenitor cells following interaction with heterodimeric receptors that share a common beta subunit required for signal transduction. Our previous studies have demonstrated that GM-CSF and IL-3 activate signaling pathways which converge upon a cAMP response element-binding protein (CREB)-binding site of the human immediate early response gene (early growth response gene-1, egr-1) promoter. Using electromobility supershift assays and antibodies directed against CREB phosphorylated on serine 133, we show that CREB is phosphorylated on serine 133 in response to GM-CSF or IL-3 stimulation. We demonstrate that phosphorylation of CREB on serine 133 substantially contributes to transcriptional activation of egr-1 in response to GM-CSF but not IL-3. These studies suggest that phosphorylation of CREB may play different roles during signal transduction, resulting in unique and overlapping biological functions in myeloid cells.