Human embryonic stem cells (hESCs) have been derived from the inner cell mass (ICM) of day 5-7 blastocysts and hold great promise for research into human developmental biology and the development of cell therapies for the treatment of human diseases. We report here that our novel three-step culture conditions successfully support the development of day-8 human blastocysts, which possess significantly (p <.01) more ICM cells than day-6 blastocysts. Plating of ICMs isolated from day-8 blastocysts resulted in the formation of a colony with hESC morphology from which a new hESC line (hES-NCL1) was derived. Our stem cell line is characterized by the expression of specific cell surface and gene markers: GTCM-2, TG343, TRA1-60, SSEA-4, alkaline phosphatase, OCT-4, NANOG, and REX-1. Cytogenetic analysis of the hESCs revealed that hES-NCL1 line has a normal female (46, XX) karyotype. The pluripotency of the cell line was confirmed by the formation of teratomas after injection into severely combined immunodeficient mice and spontaneous differentiation under in vitro conditions.