Abstract The 5T4 oncofoetal antigen is a cell surface glycoprotein that is transiently expressed during mouse ES cell differentiation and correlates with decreased pluripotency of such cells. We show that 5T4 antigen is transiently unregulated during HES4 and H1 human ES cell differentiation and its expression correlates with loss of the pluripotent markers OCT-4 and Tra-1–60 and upregulation of transcript markers associated with the three primary germ layers. To confirm that absence of cell surface 5T4 antigen represents a pluripotent hES cell phenotype, we performed mechanical transfer of either 5T4−ve or 5T4+ve HES4 colonies identified using live cell staining. 5T4−ve transfers maintained expression of OCT-4 in over 90% of resultant colonies, whereas 5T4+ve transfers exhibited significantly lower numbers of OCT-4-expressing colonies (92 ± 1.4 vs. 2.9 ± 2.0%). Interestingly, low cell density 5T4−ve colony transfers exhibited increased numbers of OCT-4-expressing colonies compared to large 5T4−ve transfers (92 ± 1.4 vs. 63.2 ± 1.9%). 5T4−ve and 5T4+ve HES4 and H1 ES cell lines expressed markers representative of neuroectoderm lineages, and we assessed the formation of neural lineages from these phenotypes in serum-containing medium and N2B27 medium. Expression of 5T4 was found to be inversely related to the yield of tyrosine-hydroxylase (TH+)-expressing neurons in N2B27 medium, with additional mesoderm and endoderm transcript markers detected. Homogeneous glial cell populations were derived from low cell density 5T4−ve colony transfers cultured in serum-containing medium, with TH+ neuronal formation inhibited in a cell-density-dependent manner. We conclude that the 5T4 antigen is a transient marker of hES cell differentiation and that 5T4 phenotype, colony seeding density and culture conditions significantly influence the maintenance of pluripotent hES cells and their differentiation to neural lineages.