Mouse AtlasTM cDNA Arrays were used for comparative studies of gene expression in mouse embryonic fibroblasts (MEF) before and after in vitro cultivation and 24 types of differentially expressed genes were identified. To screen and identify the differentially expressed new genes of MEF, cDNA suppression subtractive hybridization (SSH) technique was used. The cDNAs derived from cultured MEF after SSH were subcloned into pGEM T easy vectors to set up the subtractive library, transfected into E. coli strain JM109, sequenced and analyzed with bio informatics methods after PCR amplification.Altogether 42 types differentially expressed genes and 3 novel ESTs were identified and categorized from total 128 ESTs derived from cultured MEF after SSH. It is suggested that the genes expressed by MEF were the important molecular basis supporting the survival, proliferation and self renewal capacities of embryonic stem cells.