The current research was conducted at Vegetable and Floriculture Department in the experimental station and tissue culture laboratory, Agriculture Faculty, Mansoura University. The objective of this research was to develop a direct plant regeneration of a valuable aromatic and medicinal plant (Thymus vulgaris) by in vitro culture of shoot tips or nodal segments from mature plants on MS medium supplemented with different cytokinin type at different concentrations. Nodal segments were found to be more efficient than shoot tips for Thymus vulgaris shoots regeneration on MS medium (Murashig and Skoog 1962) supplemented with 6-benzyladenine (BA) at 2 mg/L. The best elongation was obtained on MS medium supplemented with BA at 2 mg/L in combination with GA3 at 0.5 mg/L.The best rooting of shoots was obtained on MS medium augmented with α-naphthaleneacetic acid (NAA) at 1.5 mg/L. Regenerated plants were successfully established in pots filled with mixture of soil: peat moss (1: 1 v/v). In vitro Thymus vulgaris plants had a survival rate of 100 %, and showed healthy and uniform growth.