The promise of human embryonic stem cell (hESC) lines for treating injuries and degenerative diseases, for understanding early human development, for disease modelling and for drug discovery, has brought much excitement to scientific communities as well as to the public. Although all of the lines derived worldwide share the expression of characteristic pluripotency markers, many differences are emerging between lines that may be more associated with the wide range of culture conditions in current use than the inherent genetic variation of the embryos from which embryonic stem cells were derived. Thus, the validity of many comparisons between lines published thus far is difficult to interpret. This article reviews the evidence for differences between lines, focusing on studies of pluripotency marker molecules, transcriptional profiling, genetic stability and epigenetic stability, for which there is most evidence. Recognition and assessment of environmentally induced differences will be important to facilitate the development of culture systems that maximize stability in culture and provide lines with maximal potential for safety and success in the range of possible applications.