While blastomeres of the Wortmannin 8-cell stage embryo and the cells of the ICM show some biological similarities, including a non-polarized phenotype and pluripotent potential, they also exhibit significant differences. Indeed, microarray analysis of gene expression during six developmental stages of human preimplantation embryos showed a group of 2299 probe sets differentially expressed in blastocyst stage compared to 8-cell stage embryos. The majority of these genes were involved in lipid and fatty acid metabolism. In addition, a group of 1715 probe sets involved in the regulation of transcription and nucleic acid metabolism were down-regulated in blastocysts compared to 8 cell stage embryos. Furthermore, the DNA of the blastomeres of the 8 cell stage embryos is completely demethylated as a result of reprogramming and activation of embryonic genome whereas the methylation marks are already re-established in the cells of the ICM. In addition, blastomeres of the 8-cell stage embryo exhibit lower telomerase activity and shorter average telomere length than cells in the ICM. During the derivation process, hESC acquire a polarized phenotype, a characteristic not present in the pluripotent mouse ESC. Indeed, they appear closer to mouse stem cells derived from primitive ectoderm than those from the inner cell mass. Similarly, the active molecular signaling pathways in hESC resemble those of mouse epiblast stem cells derived from postimplantation embryo. This indicates that hESC derived from the ICM acquire characteristics typical of more advanced stages of development. Hence, we investigated whether human stem cell lines derived from late totipotent blastomeres of the cleavage stage embryo develop a more “primitive” phenotype than ICM-derived lines by profiling their respective gene expression, molecular regulation and signaling pathways. In this study, we examined whether the inherent differences in the developmental stage of the starting embryonic cell population are maintained in the derived hESC lines by evaluating the stemness, differentiation capacity and gene expression profiles of blastomere- and ICM-derived hESC lines. In this study we have investigated whether differentiation status of source cells within preimplantation embryo affects phenotype and transcriptional profile of derived hESC lines. One of potential hurdles in performing such an analysis is the presence of differences between hESC cell lines that are not related to the developmental stage of the cells of origin. These differences may arise during cell line derivation and expansion as a result of in vitro selection of specific phenotypic and molecular traits/characteristics. Another potential contributing factor to hESC line heterogeneity is diverse genetic makeup of the embryos due to outbred nature of human embryos. Consequently, despite the expression of a common pluripotent phenotype, hESC lines derived from single stage embryos, namely the ICM of the blastocyst, have been reported to show significant variations in both their molecular profiles and differentiation capacity. To minimize the effect of these factors on our investigation, we used for this study multiple hESC lines with different genetic backgrounds.