Technology for the derivation, propagation, and characterization of pluripotent stem cell lines from the human embryo has undergone considerable refinement and improvement since the first published description of human embryonic stem cells in 1998. In particular, there has been extensive effort to optimize protocols and develop defined culture systems with a view toward future clinical applications of embryonic stem cell-derived products. Here, we review the current status of methodology for human embryonic stem cell derivation and culture, and we highlight the challenges that remain for workers in the field.