Induced mesenchymal stromal cells (iMSCs) derived from human pluripotent stem cells (PSCs) are attractive cells for regenerative medicine. However, the transcriptome of iMSCs and signature genes that can distinguish MSCs from fibroblasts and other cell types are rarely explored. In this study, we reported an optimized feeder-free method for the generation of iMSCs from human pluripotent stem cells. These iMSCs display a typical MSC morphology, express classic MSC markers (CD29, CD44, CD73, CD90, CD105, CD166), are negative for lymphocyte markers (CD11b, CD14, CD31, CD34, CD45, HLA-DR), and are potent for osteogenic and chondrogenic differentiation. Using genome-wide transcriptome profiling, we created an easily accessible transcriptome reference for the process of differentiating PSCs into iMSCs. The iMSC transcriptome reference revealed clear patterns in the silencing of pluripotency genes, activation of lineage commitment genes, and activation of mesenchymal genes during iMSC generation. All previously known positive and negative markers for MSCs were confirmed by our iMSC transcriptomic reference, and most importantly, gene classification and time course analysis identified 52 genes including FN1, TGFB1, TAGLN and SERPINE1, which showed significantly higher expression in MSCs (over 3 folds) than fibroblasts and other cell types. Taken together, these results provide a useful method and important resources for developing and understanding iMSCs in regenerative medicine. Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.