Mesenchymal stem cells (MSC) are self-renewing multipotent cells which hold great potential in reconstructive medicine and have been shown to be beneficial for the treatment of a variety of diseases. MSC can be derived from multiple adult tissues but have limited expansion capacity in cell culture. Highly proliferative ESC-derived MSC may be an alternative source for MSC but to date no standardized protocol exists which meets clinical standards. We extended and improved a published protocol (Raynaud et al., 2013) for the differentiation of human embryonic stem cells (ESC) into highly-proliferative MSC. To characterize the differentiation process in-depth we performed comprehensive transcriptome analysis (RNA-seq) combined with large-scale proteomic and phosphoproteomic profiling using quantitative high resolution mass spectrometric analysis. Differentiation was followed over a time course of 30 days including sampling on days 0, 1, 2, 5, 15, and 30. ESC-derived MSC were compared to adult tissue-derived MSC (bone marrow MSC) as well as to their origin (ESC). The established differentiation protocol is highly reproducible and may be adapted for clinical use. The comprehensive analysis of the differentiation process will improve understanding of MSC biology and therefore directly benefit MSC-based therapies.