Adipose-derived stem cells have been applied for the treatment of various diseases. This study was performed to fabricate the three-dimensional stem cell spheroids with adipose-derived stem cells and to evaluate the effects of cell number and culture duration on the viability and the osteogenic differentiation of three-dimensional cultures. Adipose-derived stem cells were purchased, and stem cell spheroids were formed using the silicon elastomer-based concave microwells in the amount of 1 × 105, 3 × 105, and 6 × 105. The morphology, the cell viability, and the osteogenic differentiation of stem cell spheroids were evaluated. Adipose-derived stem cells formed spheroids in the silicon elastomer-based concave microwells. A higher stem cell number led to spheroids with larger diameters. Most of the cells in the cell spheroids emitted green fluorescence. The relative Cell Counting Kit-8 showed that higher values were seen with higher stem cell numbers. The increase of alkaline phosphatase activity assays with longer incubation time in low dosage group and mineralized extracellular deposits were evenly noted in each group. This study clearly showed that adipose-derived stem cells formed spheroids in the silicon elastomer-based concave microwells and that osteogenic differentiation was achieved with higher activity and longer incubation time. Spheroid-based cell delivery could be used as a simple and effective strategy for stem cell therapy.