Stem cell migration is a critical step during the repair of damaged tissues. In order to achieve appropriate cell-based therapies for tooth and periodontal ligament repair it is necessary first to understand the dynamics of tissue-specific stem cell populations such as dental pulp stem cells (DPSC) and dental follicle stem cells (DFSC). Using time-lapse imaging, we analysed migratory and proliferative capabilities of these two human stem cell linesin vitro. When cultured alone, both DPSC and DFSC exhibited low and irregular migration profiles. In co-cultures, DFSC, but not DPSC, spectacularly increased their migration activity and velocity. DFSC rapidly surrounded the DPSC, thus resembling thein vivodevelopmental process, where follicle cells encircle both dental epithelium and pulp. Cell morphology was dependent on the culture conditions (mono-culture or co-culture) and changed over time. Regulatory genes involved in dental cell migration and differentiation such asTWIST1,MSX1,RUNX2,SFRP1andADAM28, were also evaluated in co-cultures.MSX1up-regulation indicates that DPSC and DFSC retain their odontogenic potential. However, DPSC lose their capacity to differentiate into odontoblasts in the presence of DFSC, as suggested byRUNX2up-regulation andTWIST1down-regulation. In contrast, the unchanged levels ofSFRP1expression suggest that DFSC retain their potential to form periodontal tissues even in the presence of DPSC. These findings demonstrate that stem cells behave differently according to their environment, retain their genetic memory, and compete with each other to acquire the appropriate territory. Understanding the mechanisms involved in stem cell migration may lead to new therapeutic approaches for tooth repair.