Multipotent mesenchymal stem cells (MSCs) are one of the most powerful tools in regeneration medicine. Their low differentiation efficiency, however, limits further application of MSCs in clinical therapy. Here we report that a much higher multipotent differentiation efficiency of MSCs to adult cells can be achieved using a 3D spheroid culture method based on photolithography and micropatterning techniques. MSC spheroid of precise dimension and uniform quality cultured on the microdomain substrates was prepared first, and then was induced into adipocytes and osteoblasts. Both gene expression results from RT-PCR and morphology observation results revealed that the 3D spheroid culture method could greatly improve differentiation efficiency. Gene expression profiles obtained from gene microarray analysis confirmed the high differentiation efficiency and revealed that MSCs induced in 3D spheroid culture system regulated gene expression not only by increasing the expression levels of genes related to adipogenesis and osteogenesis, but also by down-regulating the gene maintaining MSCs' self-renewal phenotypes. We conclude that our 3D spheroid culture system contributes to an optimization for efficient differentiation of MSCs, offers insight into the mechanism of efficient differentiation of engineered 3D culture system, and has promise for wide applications in regeneration medicine and drug discovery fields.