Recently, extracted teeth have been identified as a viable source of stem cells for tissue regenerative approaches. Current expansion of these cells requires incorporation of animal sera; yet, a fundamental issue underlying cell cultivation methods for cell therapy regards concerns in using animal sera. In this study, we investigated the development of a chemically defined, serum-free media (K-M) for the expansion of human periodontal ligament stem cells (PDLSCs) and human stem cells from exfoliated deciduous teeth (SHEDs). Proliferation assays were performed comparing cells in serum-containing media (FBS-M) with cells cultured in four different serum-free medium and these demonstrated that in these medium, the cell proliferation of both cell types was significantly less than the proliferation of cells in FBS-M. Additional proliferation assays were performed using pre-coated fibronectin (FN) tissue culture plates and of the four serum-free medium, only K-M enabled PDLSCs and SHEDs to proliferate at higher rates than cells cultured in FBS-M. Next, alkaline phosphatase activity showed that PDLSCs and SHEDs exhibited similar osteogenic potential whether cultured in K-M or FBS-M, and, additionally, cells retained their multipotency in K-M as seen by expression of chondrogenic and adipogenic genes, and positive Von Kossa, Alcian blue, and Oil Red O staining. Finally, differential expression of 84 stem cell associated genes revealed that for most genes, PDLSCs and SHEDs did not differ in their expression regardless of whether cultured in K-M or FBS-M. Taken together, the data suggest that K-M can support the expansion of PDLSCs and SHEDs and maintenance of their multipotency.