One of the key characteristics of human embryonic stem cells (hESC) is their ability to proliferate for an indefinite period of time. Previous studies have shown that a unique network of transcription factors are involved in hESC self renewal. Since hESC lines have the potential to differentiate into cells of all three germ layers, cells derived from hESC may be useful for the treatment of a variety of inherited or acquired diseases. The molecular signal required to differentiate hESC into a particular cell type has not been defined. It is expected that global gene expression profiling of hESC may provide an insight into the critical genes involved in maintaining pluripotency of hESC and genes that are modulated when hESCs differentiate. Several groups have utilized a variety of high throughput techniques and performed gene expression profiling of undifferentiated hESCs and mouse ES cells (mESC) to identify a set of genes uniquely expressed in ES cells but not in mature cells and defined them as “stemness” genes. These molecular techniques include DNA microarray, EST-enumeration, MPSS profiling, and SAGE. Irrespective of the molecular technique used, highly expressed genes showed similar expression pattern in several ES cell lines supporting their importance. A set of approximately 100 genes were identified, which are highly expressed in ES cells and considered to be involved in maintaining pluripotency and self renewal of ES cells. Various studies have also reported on the gene expression profiling of differentiated embryoid bodies (EB) derived from hESCs and mESCs. When hESCs are differentiated, “stemness” genes are down-regulated and a set of genes are up-regulated. Together with down-modulation of “stemness” genes and upregulation of new genes may provide a new insight into the molecular pathways of hESC differentiation and study of these genes may be useful in the characterization of differentiated cells.