The mechanisms by which hematopoietic progenitor cells become lineage-committed remain poorly understood. A cloned subline of the AML14 cell line (AML14.3D10) that spontaneously differentiates to eosinophilic myelocytes in the absence of cytokine stimulation was obtained by limiting dilution. This subline exhibits augmented expression of interleukin-5 (IL-5) receptor alpha subunit mRNA and synthesizes all major eosinophil granule proteins. Exposure of this cell line to all-trans retinoic acid (ATRA) causes loss of eosinophilic granules and fast green staining within 48 hours, without cell death. In addition, mRNA for the IL-5 receptor alpha subunit becomes undetectable by 48 hours and the cells lose responsiveness to IL-5. Major basic protein, measured as a marker of eosinophilic granule content, decreases from more than 16 pg/cell to undetectable levels by 5 days after ATRA. Concomitant with the loss of major basic protein and fast green staining, surface expression of CD16 becomes detectable and is maximum by 10 days after ATRA. mRNA for the granulocyte colony-stimulating factor (G-CSF) receptor becomes detectable by day 5, and the cells become responsive to G-CSF. At this time, the cells appear morphologically as mature neutrophils and can reduce nitroblue tetrazolium. With continued culture, the neutrophilic cells die and the culture becomes repopulated with eosinophilic myelocytes. These findings show that it is possible to change the differentiation program of hematopoietic cells even after they show evidence of advanced lineage commitment. The AML14.3D10 subclone of AML14 will be a valuable model for study of the transcriptional regulation of the eosinophil and neutrophil differentiation programs and lineage-specific gene expression.