DNA methylation plays an important role in gene silencing in mammals. The establishment of proper methylation patterns in development depends on the strict regulation of the methylation activity. The regulatory mechanism is poorly understood in development. We have shown that the two de novo methyltransferases Dnmt3a, Dnmt3b and other factors form a stable complex in mouse embryonic stem cells and function cooperatively. The two methyltransferases mutually stimulate their catalytic activity. In differentiating embryonic carcinoma, embryonic stem cells and mouse postimplantation embryos, they function synergistically to methylate the promoter of the pluripotency genes Oct4 and Nanog. Inadequate methylation caused by knocking down Dnmt3a and Dnmt3b is associated with dysregulated expression of Oct4 and Nanog in the differentiation of pluripotent cells and postimplantation embryonic development. Thus, we postulate that a physical and functional interaction between Dnmt3a and Dnmt3b represents a novel regulatory mechanism to ensure the proper establishment of genomic methylation patterns for gene silencing in development. Other potential regulatory factors including polycomb proteins are under investigation.