This chapter introduces the representative method to culture human embryonic stem cells (hESCs) under the feeder and feeder-free conditions, the former of which is used to maintain or expand undifferentiated hESCs, and the latter can be used for preparation of pure hESCs RNA samples, or for screening factors influential on self-renewal of hESCs. Also described in this chapter: a protocol and tips for conducting gene chip analysis focusing on widely used Affymetrix microarrays. These techniques will provide an unprecedented scale of biological information to illuminate a key to decipher complex signaling networks controlling pluripotency.