The clinical progression of chronic myeloid leukemia (CML) from chronic phase to blast crisis is characterized by the increasing failure of myeloid precursors to differentiate into mature granulocytes. This study was undertaken to investigate the influence of Bcr-Abl and of the small molecule Abl tyrosine–kinase inhibitor imatinib mesylate on granulocyte colony-stimulating factor (G-CSF)–induced neutrophilic differentiation. We show that differentiation of 32Dcl3 cells into mature granulocytes is accompanied by the increased expression of the antigens macrophage adhesion molecule–1 (Mac-1) and Gr-1, of the G-CSF receptor (G-CSFR), of myeloid transcription factors (CCAAT/enhancer-binding protein–α [C/EBPα], C/EBPε, and PU.1), and of the cyclin-dependent kinase inhibitor p27^Kip1. In 32Dcl3 cells transfected with thebcr-abl gene (32D_Bcr-Abl), G-CSF did not trigger either granulocytic differentiation or the up-regulation of C/EBPα, C/EBPε, and the G-CSFR. This could be correlated to a defect in c-Myc down-regulation. In contrast, the up-regulation of PU.1 and p27^Kip1 by G-CSF was not affected by Bcr-Abl. Importantly, incubation of 32D_Bcr-Ablwtcells with the kinase inhibitor imatinib mesylate prior to G-CSF stimulation completely neutralized the effects of Bcr-Abl on granulocytic differentiation and on C/EBPα and C/EBPε expression. Taken together, the results suggest that the Bcr-Abl kinase induces a reversible block of the granulocytic differentiation program in myeloid cells by disturbing regulation of hematopoietic transcription factors such as C/EBPα and C/EBPε.