OBJECTIVE.: Development of stem cell therapies for regeneration of the nucleus pulposus (NP) are hindered by the lack of specific markers to distinguish NP cells from articular chondrocytes (AC cells). This study details, for the first time, gene expression profiling to identify the human NP phenotype and assesses whether the identified markers can distinguish mesenchymal stem cell (MSC) differentiation to a correct NP cell phenotype. METHODS.: Affymetrix Microarrays were conducted on human NP and AC cells and differential expression levels for several positive (NP) and negative (AC) marker genes validated by qRT-PCR. Novel marker gene and protein expression was also assessed in human bone marrow-derived MSCs (BM-MSCs) and adipose-derived MSCs (ASCs) following differentiation in type I collagen gels. RESULTS.: Analysis identified 12 NP positive and 36 negative marker genes differentially expressed >/=20 fold and for a subset (NP positive genes PAX1, FOXF1, HBB, CA12 and OVOS2; AC genes GDF10, CYTL1, IBSP and FBLN1) differential expression was confirmed by qRT-PCR. Differentiated BM-MSCs and ASCs demonstrated significant increases in the novel NP markers PAX1 and FOXF1. ASCs lacked expression of the AC markers IBSP and FBLN1, whereas BM-MSCs lacked expression of the AC marker IBSP but expressed FBLN1. CONCLUSION.: This study has identified the phenotypic profile of human NP cells. Importantly these markers can be used to determine the in vitro differentiation of MSCs to an NP-like rather than an AC-like phenotype. Interestingly, these results suggest that ASCs may be a more appropriate cell type than BM-MSCs for IVD tissue engineering.