Spheroid models have led to an increased understanding of differentiation, tissue organization and homeostasis. In the present study, we have observed that under a serum-free medium, human dental pulp cells (DPCs) spontaneously formed spheroids, and could survive over 15 weeks. To characterize these spheroids, we investigated their dynamics, microenvironment, cell distribution, molecular profiles, and neuronal/osteogenic potential. Cell tracking assay showed that cells inside the spheroids have very slow cycling. Although the spheroids had hypoxia microenvironments, there were not any massive cell die-offs even after long-term cultivation. Whole mount immunofluorescence staining and histological analysis showed a distribution of stem cells in the central/intermediate zones of spheroids. qRT-PCR analysis demonstrated that the expression of stemness markers NANOG, TP63, and CD44 in the spheroids were much higher than within the monolayer cultures. Gene expression levels of neural markers CDH2, NFM, TUBB3, and CD24 in the spheroids were much higher than the monolayer DPCs and increased in a culture time-dependent manner. Without any neural induction, spheroid-derived cells spontaneously converted into neuron-like cells with positive staining of neural markers HuC/D and P75 under the serum-free medium for about 2 weeks. When the spheroids were transferred into osteogenic medium, they rapidly differentiated into osteo/odontogenic cells, especially the central original cells. Compared to the monolayer DPCs, mineralization in spheroids were significantly increased. This spheroid model offers a study tool to explore the molecular bases of stem cell homeostasis and tissue organization, and can be wildly used for nerve tissue and bone regeneration. J. Cell. Biochem. 114: 2624–2636, 2013. © 2013 Wiley Periodicals, Inc.