Although since 1998 more than 1,200 different hESC lines have been established worldwide, there is still a recognized interest in the establishment of new lines of hESC, particularly from HLA types and ethnic groups underrepresented among the currently available lines. The methodology of hESC derivation has evolved significantly since the initial derivations using human LIF (hLIF) for maintenance of pluripotency. However, there are still a number of alternative strategies for the different steps involved in establishing a new line of hESC. We have analyzed the different strategies/parameters used between 1998 and 2010 for the derivation of the 375 hESC lines able to form teratomas in immunocompromised mice deposited in two international stem cell registries. Here we describe some trends in the methodology for establishing hESC lines, discussing the developments in the field. Nevertheless, we describe a much greater heterogeneity of strategies for hESCs derivation than what is used for murine ESC lines, indicating that optimum conditions have not been identified yet, and thus, hESC establishment is still an evolving field of research.