Deregulation of stem cells is associated with the generation and progression of malignant tumors. In addition, genes that are associated with early embryogenesis are frequently expressed in cancer. Cripto-1 (CR-1), a glycosylphosphatidylinositol-linked glycoprotein, is expressed during early embryogenesis and in various human carcinomas. We demonstrated that human embryonal carcinoma (EC) cells are heterogeneous for CR-1 expression and consist of two distinct subpopulations: a CR-1(High) and a CR-1(Low) population. By segregating CR-1(High) and CR-1(Low) populations of NTERA2/D1 EC cells by fluorescence-activated cell sorting, we demonstrated that CR-1(High) cells were more tumorigenic than CR-1(Low) cells by an in vitro tumor sphere assay and by in vivo xenograft formation. The CR-1(High) population was enriched in mRNA expression for the pluripotent embryonic stem (ES) cell genes Oct4, Sox2, and Nanog. CR-1 expression in NTERA2/D1 cells was regulated by a Smad2/3-dependent autocrine loop, by the ES cell-related transcription factors Oct4/Nanog, and partially by the DNA methylation status of the promoter region. These results demonstrate that CR-1 expression is enriched in an undifferentiated, tumorigenic subpopulation and is regulated by key regulators of pluripotent stem cells. STEM CELLS 2010;28:1303-1314.