Dnmt1 null ESCs differentiated as embryoid bodies express high levels of trophoblast markers and, under appropriate conditions, can be differentiated into trophoblast derivatives. Similarly, it has been reported that TKO ESCs exhibit a growth defect and increased apoptosis upon EB differentiation and that cells from TKO nuclear transfer embryos aggregated with wt embryos mostly contribute to extraembryonic tissues, in addition to. However, in the latter study a few TKO cells were detected in the embryo proper till an early postgastrulation stage, but their identity was not defined. Thus, it is not clear to what extent globally hypomethylated cells are able to commit to and progress along definitive embryonic lineages. The role of DNA methylation in controlling transcription programs during mammalian development and lineage specification has been mainly inferred from genomic methylcytosine profiles in a limited selection of cell types and developmental stages, while little information is available about how DNA methylation and Dnmts actually affect transcription during differentiation. In Benzethonium Chloride particular, expression data from differentiated progeny of globally hypomethylated ESCs lacking specific Dnmts are very scarce. This is likely in relation to reports of limited survival or proliferation of Dnmt1 null and TKO cells upon differentiation. Consistent with this previous work, we found that the average size of Dnmt1 null and especially TKO EBs is reduced as compared to that of wt EBs. However, we could maintain cultures of these mutant EBs for at least 24 days. EB formation is an undirected differentiation model supporting the specification of a broad range of cell fates and thus commonly used to asses developmental potential. Using this system we found that Dnmt1 null and TKO EBs exhibit residual transcription of pluripotency master regulators Oct4 and Nanog even after 16 days of culture. However, FACS analysis showed that by day 8 Oct4 protein is uniformly downregulated in all the cells of these mutant EBs to the same basal levels as in wt EBs, suggesting homogeneous exit from the ESC state. This is supported by the relatively high concordance of genome-wide transcription changes in mutant relative to wt EBs after 4 days of differentiation, including downregulation of additional genes associated with pluripotent stem cell states and upregulation of factors related to differentiated lineages. However, by day 16 gene expression profiles in TKO EBs reveal a high degree of divergence from those in wt EBs, while expression changes in Dnmt1 null EBs are still concordant with one third of all changes in wt EBs. On the one hand this represents a previously unappreciated progression of transcription programs in differentiated Dnmt1 null cells, on the other hand it reflects substantially impaired developmental Gomisin-D potential in TKO as well as Dnmt1 null cells. In particular, ESCs lacking both PRC1 and 2 show twice the number of derepressed genes as ESCs lacking either complex, where little derepression of ERVs is observed, leading to the proposal that repetitive sequences serve as a platform for gene silencing by PRC complexes. This is reminiscent of the higher number of disregulated genes in TKO versus Dnmt1 null EBs and of substantial residual methylation of repetitive sequences in Dnmt1 null cells. It is therefore tempting to speculate that methylation of repetitive sequences may also serve as a platform for gene silencing upon differentiation.