To identify new cell surface fibroblast markers, we performed the HT-FC screen on four cultured CAF lines we had generated, as well as on five primary SOC samples, co-stained with CD45 and CD31 to allow exclusion of contaminating immune and endothelial cell types. We then selected markers that were highly expressed on the pure fibroblasts but expressed at low levels on the CD45/CD31-negative fraction of primary SOC samples, which would be expected to contain predominantly cancer cells and contaminating fibroblasts. Based on this analysis, we identified 5 candidate CAF markers. We chose to follow up on CD90, as it has been reported by others to be expressed on mesenchymal stem cells, on cells resembling mesenchymal stem cells that were cultured from SOC samples, and also to be a CAF marker in prostate cancer. Flow cytometry has long been a robust tool for reliable detection of cell surface proteins, and has many advantages over other immunological protein detection methods, such as Western blotting and IHC. These include ease of use, the ability to rapidly analyze very large cell numbers, analysis of rare populations of cells, and the ability to obtain multi-parameter information on individual cells, which is particularly important for heterogeneous cell samples. The robustness and utility of flow cytometry is illustrated by the large number of clinical applications for which it is now being used around the world. Traditionally, flow cytometry assays are performed on individual samples with a panel composed of up to 11 antibodies at a time that are known to be useful for a particular diagnosis or identification of specific cell types. More recently mass cytometry or “cytometry time-of-flight” assays, in which antibodies are labelled with lanthanide metals rather than fluorochromes and then detected by mass spectrometry, has been developed. Due to the lack of spectral overlap between antibodies, this allows an even greater degree of multiplexing to be performed, currently allowing for simultaneous analysis of up to 35 markers at a time, but with the potential to increase this up to 100 in the future. The HT-FC platform described here is complementary to these methods, as it takes a more unbiased, discovery-oriented screening approach; by screening cells of interest for a large panel of cell surface markers, it is possible to identify previously unknown proteins or protein combinations expressed on the surface of cells of interest, which once identified can then be developed into the more traditional multiplexed flow cytometry assays or translated into CyTOF assays. Thus HT-FC represents a valuable tool for new marker discovery, comparable to other discovery platforms such as gene BAY-60-7550 expression microarrays, but with a focus on cell surface proteins. The cost per sample is similar to or less than gene expression microarrays, and is significantly less than similar commercially available cell surface protein screens. Each assay can be performed in an afternoon, and there is the potential to multiplex samples by pre-labelling with fluorescent dyes to increase throughput and reduce costs even further. Data analysis is straightforward, as gates that are set on control samples in SOC.