Cellular differentiation is accompanied by dramatic changes in chromatin structure which direct the activation of lineage-specific transcriptional programs. Structure-specific recognition protein-1 (SSRP1) is a histone chaperone which is important for chromatin-associated processes such as transcription, DNA replication and repair. Since the function of SSRP1 during cell differentiation remains unclear, we investigated its potential role in controlling lineage determination. Depletion of SSRP1 in human mesenchymal stem cells elicited lineage-specific effects by increasing expression of adipocyte-specific genes and decreasing the expression of osteoblast-specific genes. Consistent with a role in controlling lineage specification, transcriptome-wide RNA-sequencing following SSRP1 depletion and the induction of osteoblast differentiation revealed a specific decrease in the expression of genes involved in biological processes related to osteoblast differentiation. Importantly, we observed a specific downregulation of target genes of the canonical Wnt signaling pathway, which was accompanied by decreased nuclear localization of active b-catenin. Together our data uncover a previously unknown role for SSRP1 in promoting the activation of the Wnt signaling pathway activity during cellular differentiation. STEM CELLS 2016;34:1369-1376 SIGNIFICANCE STATEMENT Epigenetic regulation plays an essential role in defining lineage-specific gene expression patterns. We investigated the function of the histone chaperone SSRP1 during cellular differentiation and demonstrate its essential function in specifically directing multipotent mesenchymal stem cell differentiation to the osteoblast lineage via regulation of Wnt signaling. We thereby provide the first evidence of a lineage-specific role of SSRP1 during cellular differentiation and uncover molecular insights into its function, which may serve as a basis for future clinical application of small molecule modulators of its activity for the treatment of aging-related osteoporosis and other diseases associated with altered Wnt signaling.