Human embryonic stem cell (hESC) lines have been derived and cultured in variable conditions. The idea behind derivation of hESC lines is to use them in human cell transplantation after differentiation, but already now these cells are widely used for research purposes. Despite similarities among the established lines, important differences have been reported between them, and it has been difficult to compare the results obtained using different lines. Recent optimization of hESC culture conditions has moved from cultures on mouse embryonic fibroblasts (MEFs) in fetal bovine serum-containing medium towards feeder-free culture methods using more defined animal substance-free cultures. The aim has been to establish robust and cost-effective systems for culturing these cells and eliminate the risk of infection transmitted by animal pathogens and immunoreactions caused by animal substances in cell cultures before clinical treatment. It is important to take these modifications into account when carrying out research using these cells. It is known that culture conditions influence gene expression and, hence, probably many properties of the cells. Optimization and standardization of culture methods is needed for research as well as for clinical purposes.