miR-17∼92, miR-106b∼25, and miR-106a∼363 belong to a family of highly conserved miRNA clusters. Amplification and overexpression of miR-17∼92 is observed in human cancers, and its oncogenic properties have been confirmed in a mouse model of B cell lymphoma. Here we show that mice deficient for miR-17∼92 die shortly after birth with lung hypoplasia and a ventricular septal defect. The miR-17∼92 cluster is also essential for B cell development. Absence of miR-17∼92 leads to increased levels of the proapoptotic protein Bim and inhibits B cell development at the pro-B to pre-B transition. Furthermore, while ablation of miR-106b∼25 or miR-106a∼363 has no obvious phenotypic consequences, compound mutant embryos lacking both miR-106b∼25 and miR-17∼92 die at midgestation. These results provide key insights into the physiologic functions of this family of microRNAs and suggest a link between the oncogenic properties of miR-17∼92 and its functions during B lymphopoiesis and lung development.