Embryonic stem (ES) cells have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. Human ES (hES) cell lines have been derived in multiple laboratories throughout the world. These cell populations grow as tightly compacted colonies of undifferentiated cells on mouse or human feeders or as colonies in feeder-free conditions using matrix and conditioned medium. Although mouse ES cells can be maintained in culture conditions in which the feeder layer is replaced by leukemia inhibitory factor (LIF), human cells do not appear to have this response to LIF, indicating that mouse ES cells and hES cells require different signals to maintain pluripotency. Results of two studies confirm that there are many differences between mouse and hES cells, and demonstrate the need to use multiple methodologies to characterize these differences. To date, hES cell lines have been characterized using surface markers and molecular markers used to characterize human embryonal carcinoma (EC) cells, mouse ES cells, and hematopoietic stem cells. Similar to mouse ES cells, the hES cells express alkaline phosphatase-related antigens.