In October 2003, the National Institutes of Health (NIH) established three extramural “Exploratory Centers for Human Embryonic Stem Cell Research.” Our Center’s experience in acquiring and manipulating NIH-approved human embryonic stem (hES) cell lines may be useful for other institutions interested in pursuing NIH-funded hES cell research. We acquired 14 of the 22 NIH-approved cell lines. Modifications to the proposed Material Transfer Agreements (MTAs) with hES cell suppliers were sought to improve accessibility to these hES cell lines by local researchers. Lines were characterized for survival following cryopreservation and for their ability to adapt to a uniform set of culture conditions while maintaining a normal karyotype. Each of the 4 suppliers contacted eventually agreed to terms that improved access to their hES cell lines. Eleven hES cell lines were received frozen, and in 10 cases very few cells survived cryopreservation. Ten hES cell lines were successfully converted to simplified culture conditions that enhanced their ability to be maintained and expanded in culture. One hES cell line had an unstable karyotype at an early passage. Current MTA provisions continue to present significant obstacles to NIH-funded hES cell research. Many hES cell lines can be maintained using culture conditions less onerous than those recommended by their suppliers.