Stage-specific alternative first exon (SAFE) usage is an important alternative splicing type which implicates in the regulation of many important biological processes, especially in a spatial and temporal manner. Yet the presence of SAFE transcripts and their roles in embryonic stem cells (ESCs) are still largely unknown. By comparing transcriptomes of mouse ESCs (mESCs) and somatic cells, we identify 137 mESC SAFE isoforms of 128 genes with broad expression in both ESCs and somatic cells. More than half of the mESC SAFE isoforms have open reading frame (ORF) changes as compared to the corresponding commonly expressed isoform of the same gene. The promoter regions of SAFE isoforms exhibit enriched H3K4me3 and Pol II binding as well as higher DNase I sensitivity in mESCs, but not in mouse embryonic fibroblasts (MEFs) and other somatic cells, in support of the ESC-specific expression patterns of these transcripts. Promoter regions of about 42% SAFE isoforms have interactions with key pluripotent factors Oct4, Sox2 or Nanog. Knocking-down these pluripotent related genes indeed impairs the expression of SAFE isoforms. The expression of SAFE isoforms is activated during the reprogramming process of induced pluripotent stem (iPS) cells, and dynamically regulated in early stage embryos or during cell differentiation. These results reveal the wide presence of SAFE isoforms in ESCs, the involvement of pluripotent factors in the expression regulation of SAFE isoforms indicates their functional importance in ESCs.