Human embryonic stem cells (hESCs) represent a population of undifferentiated cells with both self renewal and multilineage differentiation characteristics representing all three embryonic germ layes thus revealing their pluriptent potential. Human ESCs originate from chromosomally euploid, aneuploid, and mutant human embryos that are available from in vitro fertilization clinics treating patients for infertility or preimplantation genetic diagnosis. The pluripoent nature of hESCs has attracted great interest in using them as an important source for functional genomics, drug screening, and, perhaps eventually, cell and gene therapy. The derivation of the first hESCs was reported in 1998. Since then we have learnt a great deal about how to isolate and culture these cells to use them for the aforementioned objectives. This review gives an verview of the different methods of establishment, characterization, and maintenance of hESCs.