Nanog, Oct4 and Sox2 are important transcription factors that are expressed in embryonic stem (ES) cells or embryoniccarcinoma (EC) cells, but in most cases they are absent in somatic cells. These factors play a key role to maintain embryonic stemcell self-renew and pluripotency. Down-regulation of Nanog and Sox2 gene expression can change multiple gene expressionpatterns and signal transduction pathways, and will initiate ES cell differentiation. This study was designed to select theefficient small interfering RNA (siRNA) fragments that inhibit Nanog and Sox2 gene expression in mouse J1 ES cells and P19 ECcells. Among synthesized siRNAs we screened out the siRNA N301 for Nanog and siRNA S720 for Sox2, which not only downregulatedof Nanog and Sox2 gene expression, but also interfered embryoid bodies formation. Our study provided the definedsiRNA fragments that could be used to investigate the epigenetic function of Nanog and Sox2 genes.