Mesenchymal progenitor cells have great therapeutic potential, yet incomplete characterisation of their cell surface interface limits their clinical exploitation. We have employed subcellular fractionation with quantitative discovery proteomics to define the cell surface interface proteome of human bone marrow mesenchymal stromal/stem cells (MSCs) and human umbilical cord perivascular cells (HUCPVCs). We compared cell surface-enriched fractions from MSCs and HUCPVCs (3 donors each) with adult mesenchymal fibroblasts using 8-channel isobaric-tagging mass spectrometry, yielding relative quantification on >6000 proteins with high confidence. This approach identified 186 up-regulated proteins as mesenchymal progenitor biomarkers. Validation of 10 of these cell surface markers, including ROR2, EPHA2and PLXNA2, confirmed up-regulated expression in mesenchymal progenitor populations and distinct roles in progenitor cell proliferation, migration and differentiation. Thus cell surface protein enrichment plus isobaric-tag labelling prior to MS has delivered a comprehensive cell surface proteome repository that now enables improved selection and functional characterisation of human mesenchymal progenitor populations.