Whole tooth bioengineering has been achieved in mice using non-cultured embryonic dental mesenchymal cells (DMCs). In vitro expanded postnatal DMCs can also contribute to tooth formation but only as cells that respond to inductive signals. Since large cell numbers are required for clinical application, enabling in vitro expanded cell populations to induce de novo odontogenesis is the main challenge. To investigate if cultured DMCs can contribute to tooth formation as inductive cells, genetically-labelled cultured embryonic or postnatal DMCs were mixed with wild-type fresh inductive embryonic DMCs in different ratios, and then recombined with responsive epithelium. It is shown that although unable to initiate, cultured embryonic DMCs do not lose their ability to contribute to tooth induction; while postnatal pulp cells can neither initiate nor contribute. Microenvironmental modification of proliferating embryonic DMCs via 3D culture and co-culture systems were attempted to restore or retain the odontogenic induction ability. Although with increased expression of odontogenic genes Pax9 and Msx1, 3D cultured and co-cultured cells failed to initiate odontogenesis. Embryonic DMCs cultured for short periods (within 48 hours) maintained tooth inducing ability. Gene expression profiling was carried out with RNA sequencing data of cultured DMCs collected at different time points to study the molecular basis of the progressively reduced inductivity. In combination with gene expression comparisons between epithelial odontogenic and non-odontogenic tissues, the Wnt signalling pathway and Dlk1 were found to have a strong correlation with maintenance of odontogenic induction capacity.