The potential to generate functional pancreatic beta cells from human embryonic stem cells provides a promising avenue for beta cell replacement therapy for treatment of diabetes mellitus. Despite the rapid advancements in the field, current protocols still do not produce fully functional beta cells and the production of these cells takes at least six weeks. Understanding the molecular cues that regulate how a beta cell develops and matures would be critical in improving current approaches to generate functional beta cells. In our lab, we found that hundreds of genes enriched in functional beta cells were not properly induced in the nonfunctional cells derived from embryonic stem cells, suggesting that manipulating critical regulators that can affect multiple beta cell genes simultaneously might be instrumental in directing beta cell differentiation and maturation in the culture dish. Our studies have identified several novel regulators of gene expression, including transcription factors and small non-coding RNAs, which we predict will have critical roles in beta cell maturation. These novel regulators are highly expressed in insulin-producing islets and have very low expression in non-functional beta-like cells. This suggests that forcing expression of these regulators could accelerate the formation of functional mature beta cells. Preliminary studies so far show that forced expression of select candidates in immature precursor cells can induce the expression of several genes critical for beta cell maturation. Over the next year, we will determine, by manipulating the expression of our candidate regulators, the optimal conditions required for inducing the production of mature beta cells in vitro.