MicroRNAs (miRNAs) constitute a large family of small noncoding RNAs that have emerged as key posttranscriptional regulators in a wide variety of organisms. Because any one miRNA can potentially regulate expression of a distinct set of genes, differential miRNA expression can shape the repertoire of proteins that are actually expressed during development and differentiation or disease. Here, we have used mast cells as a model to investigate the role of miRNAs in differentiated innate immune cells and found that miR-221–222 are significantly up-regulated upon mast cell activation. Using both bioinformatics and experimental approaches, we identified some signaling pathways, transcription factors, and potential cis-regulatory regions that control miR-221–222 transcription. Overexpression of miR-221–222 in a model mast cell line perturbed cell morphology and cell cycle regulation without altering viability. While in stimulated cells miR-221–222 partially counteracted expression of the cell-cycle inhibitor p27 kip1, we found that in the mouse alternative splicing results in two p27 kip1 mRNA isoforms that differ in their 3′ untranslated region, only one of which is subject to miR-221–222 regulation. Additionally, transgenic expression of miR-221–222 from bacterial artificial chromosome clones in embryonic stem cells dramatically reduced cell proliferation and severely impaired their accumulation. Our study provides further insights on miR-221–222 transcriptional regulation as well as evidences that miR-221–222 regulate cell cycle checkpoints in mast cells in response to acute activation stimuli.