Perfluorinated compounds have raised concern due to their potential association with detrimental postnatal outcomes in animals and humans. We tested the effects of perfluorooctane sulfonate (PFOS) on a human pluripotent teratocarcinoma (known as NCCIT) cells as an in vitro prototype for developmental toxicity in mammals. NCCIT contains stem-cells able to differentiate into endoderm, mesoderm and ectoderm. We tested our cell model using a teratogenic compound, retinoic acid (RA), a cytotoxin, nocodazole (ND), and PFOS. We assayed cells proliferation, morphology and expression of stem cell and germ layer marker genes. PFOS reduced NCCIT cell proliferation in a concentration-dependent manner and induced morphological changes in cell cultures that resembled ectodermal phenotypes. A tendency towards a differentiated state in NCCIT was confirmed by real-time gene expression. PFOS triggered up-regulation of the gene nestin, indicative of ectodermal lineage differentiation, and interfered with the expression of the pluripotency stem-cell marker TERT. PFOS produced effects on both cells proliferation and differentiation, although not as severe as those observed for RA and ND, at levels that fall within the range of concentrations found in animal and human plasma. We discuss our findings in the context of possible interference of PFOS with the processes governing the early development of mammalian tissues.