Background: Odontogenisis is a complex process controlled by dynamic and reciprocal interactions that regulated by various molecules. Thymosin β4 is a small bioactive peptide with wide spectrum biological effects on much cell types. The present study was designed to highlight the effect of synthetic exogenous Tβ4 on developing dental tissue of the upper central teeth of rats, by histological examination and immunohistochemical evaluation of TGFβ1. Materials and method: Thirty six Albino Wister pregnant rat 18control group received intraperitoneal injection of normal saline and the others are experimental group received 50µg/300µl of Tβ4 injection. The animals were sacrificed at periods 16th and 18th day I.U.L and one day post natal, as six animals for each period. Histological and immunohistochemical evaluation for expression of TGF β1 in dental tissue of upper central teeth of the rat were done Results: In-vivo results showed that experimental group had accelerated stages of tooth development with acceleration in deposition of dental hard tissue (enamel and dentin) with high positive expression of TGF β1by enamel organ, dental papilla and dental sac cells. Conclusion: these data suggest synthetic exogenous Tβ4 act as bioactive initiator enhances tooth development by stimulating proliferation and differentiation of both epithelial and mesenchymal cells.