Owing to their unique ability to self-renew indefinitely, as well as their capacity to differentiate into multiple cell types of all three germ layers, embryonic stem (ES) cells hold great promise both as therapeutic agents in the clinic and as research tools in the lab. One of the main challenges in the field is understanding how stem cells achieve their remarkable potential. Recent efforts by us and others have shown that chromatin itself serves as a major contributor to ES cell identity and plasticity. While most of the supporting data emerges from biochemical and molecular studies, I propose here to use live cell imaging techniques and advanced microscopy to probe the transcriptional machinery and chromatin dynamics in living differentiating ES cells. I aim to elucidate the dynamic changes that occur in chromatin structure and function during early ES cell differentiation events. Using photobleaching methods (i.e. FRAP) complemented by biochemical approaches, I will study the dynamic interplay of both transcriptional activators (e.g. Oct-4, Nanog) and transcriptional repressors (e.g. Polycomb Group proteins) with chromatin. Also, using the spinning disk confocal technology I will monitor in real time, changes in chromatin structure, as well as the dynamics of chromatin-protein interactions in living cells. Finally, using ES cell lines carrying a labeled genomic locus at random sites I will be able to directly visualize chromatin motion in living cells. These experiments will provide new insights into the mechanisms that govern chromatin-regulated differentiation events and chromatin dynamics in living cells as well as stem cell identity and pluripotency.