The transitions from egg to embryo to adult require that cells communicate with each other to acquire specialized fates at the right times in the correct places. Cancer is in part the result of aberrant regulation. Most of our information on signaling pathways comes from genetics and biochemistry on populations of cells, with stimuli typically reduced to a binary on/off. This project will adapt contemporary microfluidic technology to accurately control the concentrations of signaling molecules in time in multiple small chambers while continuously imaging the response of single cells via fluorescently tagged proteins that move in response to signals. We will use human embryonic stem cells (hESC) for this purpose because of their relevance to regenerative medicine. We will stimulate the cells with ligands from the TGF pathway since they can induce the first round of developmental choices these cells naturally make. This pathway has two sub-branches that interfere, making it a natural context in which to study pathway interactions. Our data will be organized and developed into a predictive tool with which to rationally reprogram specialized fates from hESCs.