It is one of the core questions that how human embryonic stem cell (hESC) maintain the undifferentiated state during the propagation process. The current advance in answering this question included the studies on LIF signal transduction, Oct-4 factor and Nanog gene. But the question is far from being elucidated. In order to further understand the mechanism of maintaining the undifferentiated state of hESC, we screened out the expressed sequence tags (ESTs) which expressed highly in hESC but expressed lowly or unexpressed in the differentiated hESC's clones (dhESC) by suppression subtractive hybridization (SSH) and cDNA dot blot techniques. One hundred and five putative positive clones were screened out to be sequenced and were compared with the GenBank. Among them, seventy six clones represented sixty one identified genes that shared high homology with sequences in GenBank. Eighteen clones represented fifteen hypothetic genes, and eleven clones were function-unknown. Eight clones were picked out to analyze using semi-quantitative RT-PCR, the results confirmed that the expression of seven clones among them were higher in the hESC, and lower or no expression in the dhESC. The results showed that many differentiated expressed genes were involved before and after hESC's differentiation. Further functional analysis to those genes that expressed highly in the undifferentiated hESC and lowly in the dhESC might provide a basis to understand the mechanism of maintaining the undifferentiated state of hESC.