Cell surface markers for isolating proliferative human dental pulp stromal cells are currently lacking. Other tissues containing mesenchymal stromal cells have been studied in greater depth and candidate markers for cell isolation identified, one such marker being CD271. Previous reports suggest CD271 as a marker for isolating dental pulp stromal cells from rat incisors. We aimed to study the utility of CD271 as a marker for isolating human dental pulp stromal cells. CD271 positive cells from both third molar dental pulp and bone marrow mononuclear cells were isolated by magnetic separation followed by in vitro expansion. Phenotypic analysis was performed by flow cytometry. Our data showed that although CD271 is present in the adult molar dental pulp, significantly greater numbers of colonies are produced from the CD271 negative fraction. The opposite was seen with bone marrow mononuclear cells; all colony forming cells were derived from the CD271 positive fraction. Phenotypic analysis of expanded CD271 negative cells showed that these cells are identical to dental pulp stromal cells isolated by non-selective plastic adherence. We conclude that, in contrast to bone marrow, CD271 is not a positive selection marker for the predominant colony forming cell types from human molar dental pulp.