Maehr, P. the cell cycle on differentiation potential and Stigmasterol (Stigmasterin) present an additional perspective. hESC and hiPSC lines have a cell cycle structure characterized by an abbreviated G1 gap phase and minimal checkpoint controls3-6. In early development, the embryonic cell cycle also has a truncated G1 phase during the period when rapid cell division occurs and decisions about fate and differentiation are held back7-9. Those studies suggest that the absence of an early G1 phase promotes self-renewal, and the presence of this phase is associated with differentiation and cell fate changes. This led us to investigate whether the presence of an early G1 phase and its associated checkpoint controls are important for directed differentiation of pluripotent cell lines. We show that culturing pluripotent stem cells in dimethylsulfoxide (DMSO) activates the retinoblastoma (Rb) protein (a regulator of the G1 restriction point)7,9,10 and enhances the proportion of early G1 cells. We then show that DMSO overcomes previously reported restrictions on multilineage differentiation potential. In more than 25 different hESC and hiPSC lines, DMSO treatment increases the competency of ER81 pluripotent stem cells to respond to differentiation signals, enhances differentiation across Stigmasterol (Stigmasterin) all germ layers, and improves terminal differentiation into functional derivatives. This method permits differentiation of many cell lines toward a desired lineage and improves the prospects of using patient-specific iPSCs for disease modeling and autologous cell replacement therapy. We began our analysis by investigating the hESC line HUES8. HUES8 has one of the highest propensities for differentiation to Sox17+ definitive endoderm cells1,11, yet differentiation is not consistently high. By varying the initial plating density, we observed that the percent of cells that differentiate into definitive endoderm can range from 25% to 80% (Supplementary Fig. 1a-b), with the number of Sox17+ cells varying by as much as 7-fold (Supplementary Fig. 1c). Thus, cells are more responsive to differentiation signals if the differentiation protocol begins with cells plated at a high density. Since high density cultures are associated with increased contact-mediated growth inhibition and pluripotent stem cells have minimal sensitivity to contact inhibition6, we hypothesized that promoting contact-mediated growth inhibition in hESCs might improve their Stigmasterol (Stigmasterin) response to differentiation signals. In other tissue culture cell lines, culturing cells in DMSO can enhance contact inhibition and reversibly arrest cells in early G1 of the cell cycle12-15. Since responsiveness to differentiation signals is differentially regulated by density in HUES8 cultures, we assessed the effects of DMSO treatment on the differentiation potential of low and high density HUES8 cultures. HUES8 cultures were treated with 1% or 2% DMSO for 24 h and subsequently induced to differentiate into definitive endoderm. In low density cultures, this brief exposure to DMSO doubled responsiveness to differentiation signals (Supplementary Fig. 1d-e), increasing the percent of cells that become definitive endoderm from ~25% to 50%. DMSO treatment of high density HUES8 cultures resulted in high efficiencies comparable to control cultures (Supplementary Fig. 1e). Next, we investigated whether DMSO treatment could improve the capacity to respond to differentiation signals in a cell line that has a low propensity to make definitive endoderm. Compared to HUES8, the HUES6 cell line is much less efficient at becoming endoderm even at high density1,2 (Supplementary Fig. 1f). Treatment of HUES6 cells with 2% DMSO for 24 h prior Stigmasterol (Stigmasterin) to the onset of differentiation increased the percent of cells that became definitive endoderm from ~20% to 50% (Supplementary Fig. 1g). The H1 cell line is also predicted to have one of the lowest propensities towards the endodermal germ layer2, yet DMSO treatment induced ~90%of H1.