The capacity of human embryonic stem cells (hESCs) to self-renew indefinitely in culture while retaining their ability to differentiate into all cell types suggests that they have enormous potential both in medical applications and as a research tool (Reubinoff et al., 2000; Thomson et al., 1998). Despite their immortal nature, there is a need for derivation of new hESC lines to meet emerging requirements for their use in cell replacement therapies, disease modeling, and basic research. The need to optimize the use of donated or experimentally generated embryos motivated our attempts to improve methods for the derivation of hESC lines, which have led to practical recommendations and the generation of sibling hESC lines. Following the derivation of 17 hESC lines (Cowan et al., 2004), we derived an additional 12 lines using the same method and found that the efficiency of these derivations varied greatly from experiment to experiment (see Table S1 available online). To better understand the variables that affect derivation efficiency, we explored methods for ICM isolation and systematically investigated the relationship between preimplantation biology and the timing of ESC derivation. We found that in vitrocultured human preimplantation embryos undergo major changes in morphology as well as expression of OCT4 and CDX2 from days 5–9 postfertilization. We observed a peak of derivation efficiency using day 6 preimplantation embryos, corresponding to restriction of OCT4 to the ICM and CDX2 to the trophectoderm (TE). These comparative studies have led to the derivation of 45 new hESC lines from 140 blastocysts, of which 22 cell lines are derived from sibling embryos. Global gene expression analysis of hESC lines reveals that lines derived on different days do not significantly differ from one another in transcriptional profile, but lines derived from different genetic backgrounds do significantly differ, suggesting that genetic background, rather than the timing or method of derivation, is a contributing factor in the variability observed among hESC lines. The most widely used method for hESC derivation involves either chemical or enzymatic removal of the zona pellucida, followed by isolation of the inner cell mass (ICM) of the blastocyst by immunosurgery (Reubinoff et al., 2000; Thomson et al., 1998). In immunosurgery, cells of the TE are destroyed by brief exposure to antibodies directed against human cells in tandem with complement activity (Solter and Knowles, 1975). However, only highquality embryos with an intact TE can be subjected to this procedure, as only the structural integrity of the blastocyst prevents the ICM from also being destroyed. We reasoned that isolation of the ICM by laser-mediated ablation of the zona pellucida and TE might reduce exposure of the ICM to potentially cytotoxic compounds. The 584 frozen human embryos used in this study were donated for research following informed consent under protocols reviewed and approved by both the Committee on the Use of Human Subjects (IRB) and the Embryonic Stem Cell Research Oversight Committee (ESCRO) at Harvard University. Human zygotes and cleavage-stage embryos were thawed and cultured to the blastocyst stage (Figure S1 A). ICM isolation was carried out by exposing TE cells to celllethal laser pulses from a XYClone laser (see also Turetsky et al., 2008) and subsequent removal of dead TE cells either by using piezo drill-assisted micromanipulation or by repeated aspiration into a 50–75 mm glass capillary pipette (Figures S1 B and S1C). The isolated ICM (Figure S1 D) was then plated onto g-irradiated mouse embryonic fibroblasts (MEFs) in hESC-conditioned …