BACKGROUND: MicroRNAs are short (∼22 nt) non-coding regulatory RNAs that control gene expression at the post-transcriptional level. Here the functional impact of microRNAs on cell cycle arrest during neuronal lineage differentiation of unrestricted somatic stem cells from human cord blood (USSC) was analyzed. METHODOLOGY/PRINCIPAL FINDINGS: Expression profiling revealed downregulation of microRNAs miR-17, -20a, and -106b in USSC differentiated into neuronal lineage but not in USSC differentiated into osteogenic lineage. Transfection experiments followed by Ki67 immunostainings demonstrated that each of these microRNAs was able to promote proliferation of native USSC and to prevent in part cell cycle arrest during neuronal lineage differentiation of USSC. Bioinformatic target gene predictions followed by experimental target gene validations revealed that miR-17, -20a, and -106b act in a common manner by downregulating an overlapping set of target genes mostly involved in regulation and execution of G(1)/S transition. Pro-proliferative target genes cyclinD1 (CCND1) and E2F1 as well as anti-proliferative targets CDKN1A (p21), PTEN, RB1, RBL1 (p107), RBL2 (p130) were shown as common targets for miR-17, -20a, and -106b. Furthermore, these microRNAs also downregulate WEE1 which is involved in G(2)/M transition. Most strikingly, miR-17, -20a, and -106b were found to promote cell proliferation by increasing the intracellular activity of E2F transcription factors, despite the fact that miR-17, -20a, and -106b directly target the transcripts that encode for this protein family. CONCLUSIONS/SIGNIFICANCE: Mir-17, -20a, and -106b downregulate a common set of pro- and anti-proliferative target genes to impact cell cycle progression of USSC and increase intracellular activity of E2F transcription factors to govern G(1)/S transition.