The aim of this study was to compare the in-vitro osteogenic differentiation potential of within-subject mesenchymal stem cells (MSCs) derived from the dental pulp of permanent teeth (dental pulp stem cells - DPSCs), the dental pulp of deciduous teeth (pulp of human exfoliated deciduous teeth - SHEDs), and the periodontal ligament of permanent teeth (periodontal ligament stem cells - PDLSCs). A single subject was identified that required concurrent removal of both deciduous and permanent teeth for orthodontic purposes. Primary, mixed population cells from dental pulp, deciduous dental pulp, and periodontal ligament were obtained by the tissue out-growth method. Subsequently, isolation of STRO-1 +ve cells from their respective primary cell cultures was achieved by immunomagnetic separation. Cells were induced with an osteogenic cocktail of 5mM β-glycerophosphate, 100nM dexamethasone and 50 mg/mL ascorbic acid for up to 21 days. Osteogenic responses were assessed functionally by an alkaline phosphatase (ALP) activity assay and an alizarin red staining assay. Expression of the early osteogenic associated genes alkaline phosphatase gene (ALPL), runt-related transcription factor 2 (RUNX2), collagen type I alpha 1 (COL1A), and secreted phosphoprotein 1 (SPP1) were compared by qPCR at days 1, 4 and 7 of differentiation. Functional analysis revealed there were significant differences in intracellular ALP activity on days 4, 7, 10 & 14 with PDLSCs > SHEDs > DPSCs. Quantification of alizarin red staining showed significantly more mineralisation for PDLSCs by day 21. Gene expression analysis showed significant early up-regulations of the osteogenic markers ALPL and COL1A1 for PDLSCs over DPSCs and SHEDs. SHEDs showed significantly higher upregulation of ALPL over DPSCs. In conclusion, PDLSCs showed a significantly higher osteogenic differentiation potential than both DPSCs and SHEDs evidenced by functional studies and gene expression. This may be of significance for the use of dentally derived MSCs in bone tissue engineering applications.