Human mesenchymal stem cells (hMSCs) have the capacity to differentiate into fabricate cartilage, muscle, marrow stroma, tendon/ligament, fat, and other connective tissues, providing a potential source for tissue regeneration. The aim of this study was to find the key transcription factors (TFs), which regulated osteogenic differentiation of hMSCs. In this study, three methods were performed to find the key TFs, which included enrichment analysis, direct impact value and indirect impact value. We used the patient and public involvements (PPI) network to integrate the results of the above methods for analysis. Then, we compared the osteoblast data to the control group on days 1, 3 and 7. Finally, we found the combination of the optimal and vital 30 TFs related to osteogenic differentiation. TFs FOS, SOX9 and EP300 were commonly expressed in 3 different days in the osteogenic lineages and presented in the PPI network at relatively high degrees. Moreover, TFs CREBBP, ESR1 and EGR1 also presented high effects on the 1st, 3rd and 7th day. The constructed network gives us a more comprehensive understanding of the mechanism of osteogenesis of hMSCs.