Mesenchymal stem cells derived from the human tooth germ (hTGSCs) are a heterogeneous cell population that can differentiate into osteogenic, neurogenic, and adipogenic lineages. The aim of this study was to compare the osteogenic differentiation capacity of STRO-1 positive (STRO-1 +) hTGSCs and unsorted heterogeneous hTGSCs and to establish if STRO-1 + cells are more committed to osteogenic differentiation. HTGSCs were isolated from impacted third molar tooth germ tissues of adolescents, and a subpopulation of STRO-1 + hTGSCs was obtained by fluorescence-activated cell sorting. STRO-1 +, STRO-1 negative (STRO-1), and unsorted cells were cultured in osteogenic and standard culture media to compare their capacity to differentiate towards osteoblastic lineage. Cells were tested for proliferation rates, alkaline phosphatase activity, and amounts of accumulated calcium. Gene expression levels of the RUNX2, osteocalcin, and osteonectin genes were analyzed with real time PCR. Mineralization and osteogenic protein expression were examined by using von Kossa staining and confocal microscopy. Our results indicated that osteogenically induced cell populations showed greater mineralization capacity than non-induced cells. However, expression levels of early and late osteogenic markers were not significantly different between STRO-1 + and unsorted cells. In conclusion, the selection by STRO-1 expression does not yield cells with osteogenic capacity higher than that of the heterogeneous hTGSC population. Cell sorting using osteogenic markers other than STRO-1 might be beneficial in obtaining a more sensitive osteogenic sub-population from unsorted heterogenous hTGSCs.