The establishment of the first human embryonic stem cells (hESCs) in 1998 provided a unique tool for studying human development. Although several Western embryo-derived hESC lines are well characterized, the biological properties of Asian embryo-derived hESC lines remain unexamined. The aim of this study was to characterize Korean embryo-derived hESC lines and their differentia-tion potential. In this context, we conducted microarray-based differential gene expression analyses using two Korean embryo-derived hESC lines (CHA3 and CHA4) to identify undifferentiated and spontaneously dif-ferentiated (human embryoid body, or hEB) status. These two cell lines showed great similarity in gene expression. By comparing their expression patterns, we determined novel hESC-specific genes and transcriptomes that could serve as reliable hESC markers associated with the “stemness” phenotype. Additionally, we sought to identify hEB markers that could be used to determine the presence of differentiated cells in specific tissues, allowing for the purification of homogeneous cell populations or serving as indicators of hESC differentiation. Novel sets of 68 hESC-specific markers, 12 hESC-specific transcripts and 36 hEB markers were identified and shown by quantitative RT-PCR to be similarly expressed in CHA3- and CHA4-hESC lines, as compared to the Western embryo-derived H9-hESC line. Furthermore, our data analysis revealed that the cell cycle, urea cycle, p53 signaling, and metabolism of amino groups are significantly implicated in the regulation of hESC differentiation. These results provide another unique set of hESC/hEB markers and foster a better understanding of the molecular mechanisms underlying hESC biology. These results may thus facilitate studies of human developmental events and provide information regarding Korean embryo-derived hESCs, which could be used to determine differences in developmental events between human races.