In eukaryotic interphase cells, heterochromatin mostly localizes either at the nucleolar periphery or at the nuclear lamina. Genome localization studies are crucial due to evidence that spatial organization of the genome affects gene function. Nucleolus-associated domains (NADs) are mainly heterochromatic regions that have been mapped only in a handful of mouse and human somatic cells, and in plants. The extent to which changes in NAD localization occur during cellular differentiation remains unknown. In this thesis, we characterize a map of genome-wide NADs in F121-9 mouse embryonic stem cells (mESCs). We identified NADs by deep sequencing chromatin associated with biochemically purified nucleoli and using NADfinder software to call NAD peaks. F121-9 NADs are mostly comprised of genomic regions with inactive or lowly transcribed genes and overlap extensively with lamina-associated domains (LADs) and regions with late replication timing. Similar to somatic mouse embryonic fibroblasts (MEFs), where NADs have been previously characterized by our laboratory, F121-9 mESCs display abundant “Type I” NADs. This subset of NADs frequently associates with nuclear lamina and nucleolar periphery and resembles constitutive heterochromatin. Compared to MEFs, F121-9 mESCs have fewer “Type II” NADs; this subset of NADs is frequently found at the nucleolar periphery but not at the nuclear lamina. mESC NADs are also less enriched in H3K27me3 modified regions compared to MEF NADs. This suggests that Polycomb complex-mediated facultative vii heterochromatin expansion is part of NAD maturation during cellular differentiation. Comparison of MEF and mESC NADs also revealed enrichment of developmentally regulated genes in NADs specific to these cell types. Together, these data indicate that NADs are a developmentally dynamic component of heterochromatin. Our F121-9 mESC NAD studies identified distinct features of stem cell NADs and will facilitate future studies of genome organization changes during mammalian development.