Mammalian hematopoiesis is regulated by a complex network of genes that include both microRNAs (miRNAs) and transcription factors. miRNAs are small, 18-22 nucleotide non-coding RNAs that inhibit protein expression by targeting specific sequences in the 3' untranslated region (UTR) of their mRNA targets. We have recently demonstrated that ectopic over-expression of miRNA-125b in the bone marrow compartment of WT C57BL/6 mice causes an aggressive myeloid leukemia with increased numbers of myeloid progenitors and mature cells. Importantly, we have shown that IRF4, a tumor suppressor in myeloid cells, is a definitive target of miRNA-125b. IRF4 binds to MyD88, an adaptor protein in the NF-kB signaling pathway, to inhibit NF-kB activation. Consistent with this, ectopic over-expression of miRNA-125b or shRNA mediated knock-down of IRF4 can both lead to basal activation of macrophages and potentiation of their function. The objective of this work is to elucidate the molecular mechanism of miRNA-125b mediated myeloid disease. We hypothesize that miRNA-125b causes an aggressive myeloid disease by negatively regulating expression of IRF4, a negative regulator of NF-kB signaling. In Aim 1, we further characterize the role of miRNA-125b in myeloid development and leukemia, with an emphasis on determining the cellular mechanism of disease. In Aim 2.1, we will investigate the contribution of IRF4 to the miRNA-125b mediated leukemic phenotype. Finally, in Aim 2.2, we will study the importance of NF-kB signaling in increasing myeloid output and causing leukemia with miRNA-125b over-expression. Overall, we hope to understand the function of miRNA-125b, which is deregulated in several human cancers, in hematopoiesis in a relevant in vivo system. Importantly, we will also elucidate a set of basic principles on how a miRNA can alter the balance between normal and aberrant hematopoiesis.