The induction of pluripotency from adult cells has enormous potential in regenerative medicine. While initial efforts to study mechanisms and improve efficiency of induced pluripotent stem cell (iPSC) reprogramming focused on the direct roles of transcriptional regulators, increasing evidence indicates that cellular signal transduction pathways can modulate this process. Here, we present a medium-throughput system to study the effect of signaling pathways on the early stages of reprogramming. We generated a set of lentiviral vectors encoding 38 genes that upregulate or downregulate major signal transduction pathways and quantified each signaling factor's effect on reprogramming. This approach confirmed the role of several factors previously implicated in reprogramming, as well as identified several GTPases-factors that to date have not been largely studied in reprogramming-that improve or hinder iPSC reprogramming. In addition, this methodology is useful in determining new targets for enhancing pluripotency reprogramming, lineage reprogramming, and/or cell differentiation. (C) 2014 Wiley Periodicals, Inc.