Cultured pluripotent stem cells hold great promise for regenerative medicine. Considerable efforts have been invested into the refinement and definition of improved culture systems that sustain self-renewal and avoid differentiation of pluripotent cells in vitro. Recent studies have, however, found that the choice of culture condition has a significant impact on epigenetic profiles of cultured pluripotent cells. Mouse and human ESCs (embryonic stem cells) show substantial epigenetic differences that are dependent on the culture condition, including global changes to DNA methylation and histone modifications and, in female human ESCs, to the epigenetic process of X chromosome inactivation. Epigenetic perturbations have also been detected during culture of pre-implantation embryos; limited research undertaken in mouse suggests a direct effect of the in vitro environment on epigenetic processes in this system. Widespread epigenetic changes induced by the culture condition in stem cells thus emphasize the necessity for extensive research into both immediate and long-term epigenetic effects of embryo culture during assisted reproductive technologies.