Early hematopoietic progenitors expressing the CD34⁺ phenotype can be harvested from the peripheral blood of normal individuals. We have optimized the liquid culture of human CD34⁺ peripheral blood progenitors (PBPs) to achieve differentiation into a population of cells consisting almost entirely of eosinophil progenitors and maturing eosinophils. Growth of CD34⁺ PBPs for 28 days in the presence of the combination of IL-3, granulocyte-macrophage colony-stimulating factor, and IL-5 resulted in an almost 250-fold increase in cell number, yielding a population that contained 83% maturing eosinophils. The residual population consisted of basophils and mast cells (3% by acidic toluidine blue staining, 15.2% by flow cytometric assay for binding to high-affinity IgE receptor) and immature cells. This provides an opportunity to examine the kinetics of the acquisition of specialized mature eosinophil characteristics during eosinophil differentiation. Several host-defense and bioactive proteins are found almost exclusively in eosinophil granules. In addition, stimulated eosinophils, like neutrophils, produce copious amounts of toxic oxygen radicals. We used our culture system and the sensitive technique of reverse-transcriptase polymerase chain reaction to analyze the kinetics of production of messenger RNA transcripts encoding several eosinophil proteins, including five eosinophil granule proteins and four subunit peptides of the superoxide-generating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in small numbers of differentiating eosinophils from peripheral blood CD34⁺ cells. Freshly isolated CD34⁺ PBPs contained transcripts for the ubiquitously present housekeeping protein phosphoglucokinase but contained no eosinophil granule protein transcripts and barely detectable amounts of some oxidase protein transcripts. On day 3 of culture, no cells recognizable by histochemical staining as eosinophils could be detected, but transcripts for all five eosinophil granule proteins were present. These transcripts increased several fold during the entire culture period. Similar kinetics were seen for all but one of the NADPH oxidase protein transcripts. However, transcripts for the p67^phox NADPH oxidase protein were not detected until day 7, and functional oxidase activity did not appear until day 12. From that point, oxidase activity increased dramatically over the culture period. These studies demonstrate that commitment of CD34⁺ PBPs to the eosinophil lineage occurs very early, by day 3, but that further events in differentiation must take place before the appearance of histologically staining eosinophil granules and acquisition of functional oxidase capacity. (J ALLERGY CLIN IMMUNOL 1996;98:344-54.)