Updated project metadata. The intriThe intricate balance between MSCs differentiation to osteoblasts or adipocytes is finely regulated. To explore novel participating molecules, we screened for early-stage osteogenesis- or adipogenesis-based MSCs protein expression profile using TMT-based quantitative proteomic analysis. Protein annotation, hierarchical clustering, functional stratification, and protein-protein association assessments were performed. Moreover, two upregulated proteins, namely, FBLN2 and NPR3, were validated to participate in the osteogenic differentiation process of MSCs. Subsequently, we independently downregulated FBLN2 and NPR3 during 7 days of osteogenic differentiation, and conducted quantitative proteomics analysis to assess the differential protein regulation between knockdown and control cells. Based on gene ontology (GO) and network analyses, FBLN2 deficiency induced functional alterations associated with biological regulation and stimulus response, whereas, NPR3 deficiency induced functional alterations related to cellular and metabolic processes, and so on. These results demonstrated that proteomics is still an effective tool for the comprehensive exploration of the MSCs differentiation process. cate balance between MSCs differentiation to osteoblasts or adipocytes is finely regulated. To explore novel participating molecules, we screened for early-stage osteogenesis- or adipogenesis-based MSCs protein expression profile using TMT-based quantitative proteomic analysis. Protein annotation, hierarchical clustering, functional stratification, and protein-protein association assessments were performed. Moreover, two upregulated proteins, namely, FBLN2 and NPR3, were validated to participate in the osteogenic differentiation process of MSCs. Subsequently, we independently downregulated FBLN2 and NPR3 during 7 days of osteogenic differentiation, and conducted quantitative proteomics analysis to assess the differential protein regulation between knockdown and control cells. Based on gene ontology (GO) and network analyses, FBLN2 deficiency induced functional alterations associated with biological regulation and stimulus response, whereas, NPR3 deficiency induced functional alterations related to cellular and metabolic processes, and so on. These results demonstrated that proteomics is still an effective tool for the comprehensive exploration of the MSCs differentiation process.