Recently, microRNAs have been shown to be involved in hematopoietic cell development, but their role in eosinophilopoiesis has not yet been described. In this article, we show that miR-223 is upregulated during eosinophil differentiation in an ex vivo bone marrow-derived eosinophil culture system. Targeted ablation of miR-223 leads to an increased proliferation of eosinophil progenitors. We found upregulation of a miR-223 target gene, IGF1R, in the eosinophil progenitor cultures derived from miR-223(-/-) mice compared with miR-223(+/+) littermate controls. The increased proliferation of miR-223(-/-) eosinophil progenitors was reversed by treatment with an IGF1R inhibitor (picropodophyllin). Whole-genome microarray analysis of differentially regulated genes between miR-223(+/+) and miR-223(-/-) eosinophil progenitor cultures identified a specific enrichment in genes that regulate hematologic cell development. Indeed, miR-223(-/-) eosinophil progenitors had a delay in differentiation. Our results demonstrate that microRNAs regulate the development of eosinophils by influencing eosinophil progenitor growth and differentiation and identify a contributory role for miR-223 in this process.