The miR-17-92 cluster and its 6 encoded miRNAs are frequently amplified and aberrantly expressed in various malignancies. This study demonstrates that retroviral-mediated miR-17-92 overexpression promotes expansion of multipotent hematopoietic progenitors in mice. Cell lines derived from these miR-17-92-overexpressing mice are capable of myeloid and lymphoid lineage differentiation, and recapitulate the normal lymphoid phenotype when transplanted to nonobese diabetic/severe combined immunodeficiency mice. However, overexpression of individual miRNAs from this locus, miR-19a or miR-92a, results in B-cell hyperplasia and erythroleukemia, respectively. Coexpression of another member of this cluster miR-17, with miR-92a, abrogates miR-92a-induced erythroleukemogenesis. Accordingly, we identified several novel miR-92a and miR-17 target genes regulating erythroid survival and proliferation, including p53. Expression of this critical target results in marked growth inhibition of miR-92a erythroleukemic cells. In both murine and human leukemias, p53 inactivation contributed to the selective overexpression of oncogenic miR-92a and miR-19a, and down-regulation of tumor-suppressive miR-17. This miR-17-92 expression signature was also detected in p53- B-cell chronic lymphocytic leukemia patients displaying an aggressive clinical phenotype. These results revealed that imbalanced miR-17-92 expression, also mediated by p53, directly transforms the hematopoietic compartment. Thus examination of such miRNA expression signatures should aid in the diagnosis and treatment of cancers displaying miR-17-92 gene amplification.