Human bone tissue is built in a hierarchical way by assembling various cells of specific functions; the behaviors of these cells in vivo are sophisticatedly regulated. However, the cells in an injured bone caused by tumor or other bone-related diseases cannot properly perform self-regulation behaviors, such as specialized differentiation. To address this challenge, a simple one-step strategy for patterning drug-laden poly(lactic-co-glycolic acid) (PLGA) microspheres into grooves by Teflon chips is developed to direct cellular alignment and osteogenic commitment of adipose-derived stem cells (ADSCs) for bone regeneration. A hydrophilic model protein and a hydrophobic model drug are encapsulated into microsphere-based grooved micropatterns to investigate the release of the molecules from the PLGA matrix. Both types of molecules show a sustained release with a small initial burst during the first couple of days. Osteogenic differentiated factors are also encapsulated in the micropatterns and the effect of these factors on inducing the osteogenic differentiation of ADSCs is studied. The ADSCs on the drug-laden micropatterns show stronger osteogenic commitment in culture than those on flat PLGA film or on drug-free grooved micropatterns cultured under the same conditions. The results demonstrate that a combination of chemical and topographical cues is more effective to direct the osteogenic commitment of stem cells than either is alone. The microsphere-based groove micropatterns show potential for stem cell research and bone regenerative therapies.