We previously reported that cynomolgus monkey embryonic stem (ES) cells could be maintained under a feeder-free condition without using recombinant cytokines if sizes and numbers of ES colonies were kept within an appropriate range. Here we show that this finding is also true with human ES cells (hESCs). The two lines of hESCs, khES-1 and khES-3, were appropriately maintained in the absence of feeder layers or exogenous cytokines such as fibroblast growth factors, Noggin, transforming growth factor beta, and Activin by closely controlling the size and number of hESC colonies. High-level expressions of immature markers including SSEA-4, Oct-4, and Nanog were detected in feeder-free and cytokine-free hESCs, and they formed teratomas when implanted into severe combined immunedeficiency (SCID) mice. No chromosomal abnormalities were observed over 20 passages, ruling out the possibility that special clones with growth advantages had been selected. Global protein expression profiles were quite similar among the hESCs maintained by our feeder- and cytokine-free method, by coculture with mouse embryonic fibroblasts (MEFs) and by a feeder-free method using conditioned media of MEFs. However, the activation level of Akt, an important player for the maintenance of ES cells, was highest and the activation level of extracellular signal-regulated kinase, a critical player for differentiation of ES cells, was lowest in the hESCs maintained by our cytokine-free method. Our results not only show a technical improvement for the maintenance of hESCs but also open a new avenue for the understanding of autocrine signaling networks of hESCs.