MicroRNAs are short ribonucleic acids (RNAs) that play an important role in many aspects of cellular biology such as differentiation and apoptosis, due to their role in the regulation of gene expression. Using microRNA microarrays, we characterized the microRNA gene expression of 27 patients with acute myeloid leukemia (AML) with normal cytogenetics, focusing on the microRNAs differentially expressed between the M1 and M5 French–American–British (FAB) subtypes. An accurate delineation of these two AML entities was observed based on the expression of 12 microRNAs. We hypothesized that these microRNAs may potentially be involved in the differentiation block of M1 blasts and consequently monocytic differentiation. Using publically available mRNA data and microRNA target prediction software, we identified several key myeloid factors that may be targeted by our candidate microRNAs. The expression changes of the candidate microRNAs during monocytic differentiation of AML cell lines treated with Vitamin D and phorbol 12-myristate 13-acetate were examined. All six candidate microRNAs were significantly down-regulated over the time course by quantitative reverse transcriptase polymerase chain reaction suggesting a link between these microRNAs and monocytic differentiation. To further characterize these microRNAs, we confirmed by luciferase assays that these microRNA target several key myeloid factors such as MAFB, IRF8, and KLF4 identifying a possible mechanism for the control of differentiation by these microRNAs. Am. J. Hematol., 2011. © 2010 Wiley-Liss, Inc.