An avulsed tooth must be stored in a solution which maintains periodontal ligament (PDL) cell viability. The aim of this study was to compare the effects of Hank's balanced salt solution (HBSS) and milk on the differentiation of PDL fibroblasts. Eighteen extracted third molars, (n=3 for each group), were immersed in HBSS, milk, and Dulbecco's modified Eagle medium-Ham's F12 (DMEM-F12) at 4°C for 30- to 60 minutes or 12hours. The growth dynamics of PDL fibroblasts were evaluated with cell proliferation graphics and population doubling time (PDT) values. Runt-related transcription factor 2 (RUNX2), receptor activator of nuclear factor kappa-B ligand (RANKL), and collagen type XII (COL12) expression were used to evaluate the differentiation of PDL fibroblasts. The percentage of cell numbers and PDT values of groups were statistically insignificant. In the HBSS groups, RUNX2 expression increased showing a direction to osteogenic differentiation of PDL fibroblasts. In the DMEM-F12 groups, RANKL expression increased, indicating there was a tendency for osteoclastogenic differentiation. In the milk groups, RUNX2 expression decreased while other markers were stable showing PDL fibroblasts could protect fibroblast identity. In terms of protecting fibroblast identity and resistance to differentiation, milk was more effective than HBSS.