MicroRNA 92b controls the G1/S checkpoint gene p57 in human embryonic stem cells. Human embryonic stem (ES) cells exhibit a shorter G (1) cell cycle phase than most somatic cells. Here, we examine the role of an abundant, human ES cell-enriched microRNA, miR-92b, in cell cycle distribution. Inhibition of miR-92b in human ES cells results in a greater number of cells in the G (1) phase and a lower number in the S phase. Conversely, overexpression of miR-92b in differentiated cells results in a decreased number of cells in G1 phase and an increased number in S-phase. p57, a gene whose product inhibits G (1) to S-phase progression, is one of the predicted targets of miR-92b . Inhibition of miR-92b in human ES cells increases p57 protein levels, and miR-92b overexpression in differentiated cells decreases p57 protein levels. Furthermore, miR-92b inhibits a luciferase reporter construct that includes part of the 3' untranslated region of the p57 gene containing the predicted target of the miR-92b seed sequence. Thus, we show that the miRNA miR-92b directly downregulates protein levels of the G (1)/S checkpoint gene p57 . STEM CELLS 2009; 27: 1524-1528.