Different cues present in the cellular environment control basic biological processes. A previously established 3D microwell array was used to study dimensionality-related effects on osteogenic differentiation and plasticity of marrow stromal cells. To enable long-term culture of single cells in the array a novel surface functionalization technique was developed, using the principle of subtractive micro contact printing of fibronectin and surface passivation with a triblock-copolymer. Immunohistochemical stainings showed that when cultivated in 3D microenvironments, marrow stromal cells can be maintained in the wells for up to 7 days and be induced to commit to the osteogenic lineage. In conclusion, this work shows the modification of a 3D microwell array allowing the long term study of single stem cell plasticity and fate in controlled microenvironments.