Mouse dental papilla cells (MDPCs) can be differentiated into component cell types that have the ability to regenerate the pulpo-dentinal complex. At the cap stage of tooth development, MDPC component cells are programmed for tooth generation. Therefore, isolated and cultivated component cells can be useful in evaluating cellular, molecular, and environmental scenarios in tooth germ formation and tooth morphogenesis. Increasing the proliferation capacity of MDPCs and preserving their original phenotypic and genotypic characteristics for tooth organ regeneration are the main focus of this study. An immortalized mouse dental papilla cell line was created via the intracellular insertion of SV40 T antigens into the nucleus by lentivirus particles. The generated clonally isolated SV40 T immortalized MDPC line was then characterized and validated for transfection success and efficiency. These cells displayed a higher proliferation rate, and both genotype and phenotype characteristics were similar to those of the original primary cell line. These results were verified via the expression of a broad array of tooth-specific markers. Furthermore, the test results to show that transformed cells had preserved multi-potency were also positive. Thus, the stable immortalized MDPCs may be used to determine the mechanisms of an array of developmental phenomena, such as early dental cell proliferation, reconstitution of tooth germ, dentine mineralization and other significant growth factor signaling pathways influencing tooth morphogenesis.