Stem cell fates on biomaterials are influenced by the complex confluence of microenvironmental cues emanating from soluble growth factors, cell-to-cell contacts, and biomaterial properties. Cell-microenvironment interactions influence the cell fate by initiating a series of outside-in signaling events that traverse from the focal adhesions to the nucleus via the cytoskeleton and modulate the sub-nuclear protein organization and gene expression. Here, we report a novel imaging-based framework that highlights the spatial organization of sub-nuclear proteins, specifically the splicing factor SC-35 in the nucleoplasm, as an integrative marker to distinguish between minute differences of stem cell lineage pathways in response to stimulatory soluble factors, surface topologies, and microscale topographies. This framework involves the high resolution image acquisition of SC-35 domains and imaging-based feature extraction to obtain quantitative nuclear metrics in tandem with machine learning approaches to generate a predictive cell state classification model. The acquired SC-35 metrics led to >90% correct classification of emergent human mesenchymal stem cell (hMSC) phenotypes in populations of hMSCs exposed for merely 3 days to basal, adipogenic, or osteogenic soluble cues, as well as varying levels of dexamethasone-induced alkaline phosphatase (ALP) expression. Early osteogenic cellular responses across a series of surface patterns, fibrous scaffolds, and micropillars were also detected and classified using this imaging-based methodology. Complex cell states resulting from inhibition of RhoGTPase, β-catenin, and FAK could be classified with >90% sensitivity on the basis of differences in the SC-35 organizational metrics. This indicates that SC-35 organization is sensitively impacted by adhesion-related signaling molecules that regulate osteogenic differentiation. Our results show that diverse microenvironment cues affect different attributes of the SC-35 organizational metrics and lead to distinct emergent organizational patterns. Taken together, these studies demonstrate that the early organization of SC-35 domains could serve as a "fingerprint" of the intracellular mechanotransductive signaling that governs growth factor- and topography-responsive stem cell states.