Cellular microRNAs (miRNAs) are pivotal regulators involved in various biological processes through the post-transcriptional regulation of gene expression. Signaling pathways are extensively activated during 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced differentiation of human leukemia cells, but the modulation of miRNA expression and processing in this context has yet to be fully explored. In this study, we comprehensively analyzed 10 miRNAs that are consistently upregulated during TPA-induced differentiation of various leukemia cell lines by employing microarray technology. The upregulation of these miRNAs was further verified by quantitative RT-PCR, and, markedly, a subset of the miRNAs was found to be induced via the MEK/ERK signaling pathway using TPA and specific pharmacological inhibitors. Moreover, immunoblotting and quantitative RT-PCR analysis demonstrated that the expression levels of key miRNA processing machineries (i.e., Drosha, Dicer, Ago1 and Ago2) were not induced in this context, but the transcription of the miRNA products was triggered by MEK/ERK activation. Therefore, we identified the unique miRNAs that respond to TPA treatment in leukemia cells and demonstrated the essential role of the MEK/ERK signaling pathway in the induction of these miRNA transcripts.