Different nutrient media (MS [Murashige and Skoog 1962]; QL [Quoirin and Lepoivre 1977] and WPM [Lloyd and McCown 1980]); plant growth regulators BA (benzil adenin), GA3 (gibberellic acid), IBA (indole-3-butyric-acid), NAA (naftalen acedic acid); and sucrose concentrations were studied to determine the in vitro culture effects on healthier and faster seedling development from mature lentisk (Pistacia lentiscus L.) seeds. After 28 days of culture, the percentage of germinated seeds was the highest (70%) in the full-strength MS medium. The cytokinin BA was superior to other tested treatments in terms of its ability to promote germination of lentisk seeds. When tested at different concentrations, sucrose gave the best results obtained at concentrations of 1–4%, whereas high concentrations (6 and 8%) mainly decreased germination rate and there was no a regular pattern for elongation of the aerial parts of plants. With this described protocol, on average 76.67% seeds germinated 4 weeks after culture. Developed seedlings were satisfactorily acclimatized in sterilized peat, soil and perlite containing compost, with high percentage survival viability was obtained 9 months after transfer to in vivo conditions (93.33%). The results obtained showed that the enriched full-strength MS medium supplemented with 1 mg L–1 BA and 3% sucrose induced homogeneous and healthy seedling development in a period of 4 to 8 weeks of culture.