Embryonic stem (ES) cells are pluripotent cells isolated from mammalian preimplantation embryos. They are capable of differentiating into all cell types and therefore hold great promise in regenerative medicine. Here we show that murine ES cells can be fully SILAC-labeled when grown feeder-free during the last phase of cell culture. We fractionated the SILAC-labeled ES cell proteome by one dimensional gel electrophoresis and by isoelectric focusing of peptides. High resolution analysis on a linear ion trap-orbitrap instrument (LTQ-Orbitrap) at sub-ppm mass accuracy resulted in confident identification and quantitation of more than 5,000 distinct proteins. This is the largest quantified proteome reported to date and contains prominent stem cell markers such as Oct4, Nanog, Sox2 and Utf1 along with the embryonic form of Ras (ERas). We also quantify the proportion of the ES cell proteome present in cytosolic, nucleoplasmic and membrane/chromatin fractions. We compared two different preparation approaches – cell fractionation followed by 1D gel separation and in-solution digestion of total cell lysate combined by isoelectric focusing, and found comparable proteome coverage with no apparent bias for any functional protein classes for either approach. Bioinformatic analysis of the ES cell proteome reveals a broad distribution of cellular functions with overrepresentation of proteins involved in proliferation. We compare the proteome with a recently published map of chromatin states of promoters in ES cells and find excellent correlation between protein expression and the presence of active and repressive chromatin marks.