Embryonic stem cells (ESCs) represent a unique cell type that is characterized by the ability to self-renew and differentiate into all types of somatic cells, as well as into germ cells. An understanding of the mechanisms which underlie their functioning is necessary for their usage in regenerative medicine. Pcbp1 is a DNA/RNA poly(C)-binding protein with a broad spectrum of functions including transcription, mRNA processing, and alternative splicing. In this study, we demonstrate that Pcbp1 blocks spontaneous ESCs differentiation towards primitive endoderm (PrE) by fine-tuning MEK/ERK signaling. Knockout of Pcbp1 leads to a specific PrE Oct4+Gata6+ population which is not receptive to differentiation signals. MEK/ERK inhibition prevents this phenotype but as our RNA-seq and Mass-spec data show, further Naïve-to-Primed transition is failed and leads to cell death, which is caused by the conflict of Primed and PrE gene expression network. Our results could explain an early lethal phenotype of Pcbp1-knockout mouse embryos and make clear the affected mechanisms. This dataset represents shotgun proteomics comparison of Mus musculus ESCs in Naïve to Primed states with or without Pcbp1 mutation.