Mesenchymal stem cells (MSCs) are a type of somatic stem cells that are important in regenerative medicine due to their multipotent differentiation and therapeutic properties. To regulate their lineage commitment, use of several functional surfaces were reported. Micropatterned surfaces are very useful to regulate adhesion state and lineage commitment of MSCs with control of cell shape, cell spreading area, and the number of cell-cell contacts in 2 dimensional (2D) culture. However, there are no reports that study the change in differentiation property of MSCs by varying their 3D accumulation levels. Here, we propose the new use of micropatterned surface to control cell accumulation within a range from 2D to 3D culture and investigate cell-accumulation dependent alteration of differentiation property in adipose-derived stem cells (ADSCs). ADSCs were seeded with varied cell number onto micropatterned surface, where different accumulation series of ADSCs were constructed on circular adhesion areas with 100 µm-diameters. Structural characterization using confocal microscopy showed that this series of ADSCs had different accumulation levels in z-axis from monolayer to more than three layers. The relationship between 3D cell accumulation levels and differentiation potentials of MSCs was evaluated before differentiation induction by measuring the expression of differentiation marker genes. Osteogenic potentials were enhanced with the increase of 3D cell accumulation, while adipogenic potentials were repressed. Interestingly, in terms of cellar density, these unique responses of MSCs with 3D cell accumulation is opposite to that with 2D. These results suggest that micropatterned surface can be used to control 3D cell accumulation accurately and become a tool for regulating the lineage commitment of MSCs.