PROJECT SUMMARY The progression from a pluripotent state into the myriad cell types that arise during mammalian development involves a host of epigenetic properties that dictate gene expression patterns and enforce the commitment of a cell to a specific lineage. The advent of single-cell genomics has enabled the study of cell lineage dynamics at unprecedented resolution; however, such studies have been largely limited to RNA transcription. The dynamic modulation of the epigenome that governs these processes involves multiple layers of regulation, including a major role by DNA methylation – a canonically repressive property that can act as a gatekeeper of genomic permissiveness. Aberrant DNA methylation is also associated with numerous developmental disorders and diseases; however, it is also one of the least studied properties, particularly at the single-cell level, primarily due to technological limitations. We have recently addressed this shortcoming with the development of a high- throughput single-cell DNA methylation assay capable of producing 100’s of thousands of cell profiles in a single experiment, paving the way for studies of this property with a power comparable to that of transcription. Here we propose the production of at atlas of DNA methylation during mammalian embryonic development in the mouse at 24-hour increments from embryonic day 8.5 to 17.5 in order to produce a granular map of DNA methylation dynamics during key stages of lineage priming and commitment. We will further assess the intestinal epithelium using novel multiomic technologies in order to gain mechanistic insight into how layers of epigenetic control drive and enforce one another. These data will enable the assessment of standing hypotheses regarding the role of DNA methylation in genomic permissiveness and cell fate decisions. Finally, the datasets, which will represent a powerful resource for developmental biology and epigenomic studies, will be made openly available through interactive portals to enable broad access by the greater research community.