Stromal cells have been widely used in biomedical research and disease modeling studies in vitro. The most commonly used types of stromal cells are mesenchymal stem cells and fibroblasts. Their cellular phenotypes and differentiation capabilities are quite similar and there are no specific distinction criteria. In order to identify transcriptomic differences between these 2 cell types, we performed next-generation RNA sequencing. Using the global gene expression profile and pathway analysis, we showed that human primary bone marrow mesenchymal stem cells and human primary dermal fibroblasts have different molecular signatures. We also identified critical transcription factors that are differentially expressed between these cells. We then proposed that homeobox genes and some other sequence-specific transcription factors are not only responsible for transcriptional differences, but also discriminate bone marrow mesenchymal stem cells and dermal fibroblasts at the transcriptional level.