Potato (Solanum tuberosum L.) is an economic tuberous crop cultivated worldwide in the temperate, tropical and subtropical zones. It occupies the fourth largest food crop following wheat, rice and maize. The aim of this study is to establish a protocol for in vitro initiation, multiplication, rooting and ex vitro acclimatization of potato plants (Solanum tuberosum L.) cultivars Lady Balfour and Bellini. This study was carried out in the plant tissue culture laboratory, the Faculty of Agriculture, Saba basha, Alexandria University, Egypt during the period from 2013 to 2016. An efficient and reliable protocol for in vitro propagation and ex vitro acclimatization of potato (Solanum tuberosum L.) was optimized. Nodal cutting explants were inoculated on various initiation or establishment media with different combinations of NAA and KIN and the neoformed shoots were cultured on proliferation (multiplication) media with different combinations of NAA and BAP for the development of multiple shoots, and the elongation media to elongate of the neoformed shoot. The subsequent elongated shoots were rooted, and acclimatized     ex vitro, successfully. The best medium for shoot initiation was MS medium supplemented with 1.0 mg/l KIN. The favorable medium for multiplication was the tested medium augmented with 2.0 mg/l BA and 0.250 mg/l NAA. In addition, the most effective medium for elongation was the used medium enriched with 0.250 mg/l NAA. Furthermore, in vitro the shoots showed healthy root development when the tested medium was supplemented with  combination of 1.0 mg/l IBA and 0.50 mg/l NAA (rooting stage).The combination of sand:perlite:peatmoss (1:3:3, v:v:v) was used as substrates for the hardening of the in vitro plantlets, as a potting mix, was the best suited mix for the acclimatization of plantlets ex vitro.