The purpose of this study was to produce a metabolomic profile of a human mesenchymal stem cell (hMSC) pellet sample to identify biomarkers of adipogenic differentiation using nuclear magnetic resonance (NMR) spectroscopy. hMSC adipogenesis was monitored over four cycles of differentiation; one cycle consisted of treatment with adipogenic induction medium for 3 days followed by adipogenic maintenance medium for 1 day. Adipogenesis in control hMSCs was confirmed by Oil Red O staining. A NMR micro-imaging machine with a zqpr pulse (total volume) sequence was used to examine the metabolic changes in the cell pellet samples. Several lipid peaks were predominant among the various metabolite peaks in the NMR spectroscopy data. In particular, the lipid methylene (-[CH2]n-) signal at 1.3 ppm increased 3.6-fold during the first cycle, 15.7-fold during the second cycle, and 28.3-fold during the third cycle. Our findings indicate that NMR spectral peaks related to lipid metabolites can be used as a biomarker of hMSC adipogenesis.