Cell suspension cultures of Arnebia euchroma were raised from in vitro leaf-derived friable callus on liquid MS [Murashige and Skoog] medium supplemented with BAP (6-benzylaminopurine) (10.0 μM) and IBA (indole-3-butyric acid) (5.0 μM). A two-stage culture system was employed using growth and production medium for cell biomass and shikonin derivatives, respectively. Factors such as light, temperature, sucrose and pH (hydrogen ion concentration) were studied to observe their effect on the shikonin derivative production. Light conditions completely inhibited shikonin derivative production. Out of different temperature regimes tested, the highest yield (586.17 μg/g FW) was found at 25°C. Maximum production (656.14 μg/g FW) was observed in 6% sucrose. An alkaline pH (7.25-9.50) favoured shikonin derivative production. The results showed that physical and chemical factors greatly influence the production of shikonin derivatives in cell suspension cultures of A. euchroma. Therefore, by employing optimum culture conditions, it is possible to enhance the production of secondary compounds from the cells. The factors optimized for in vitro production of shikonin derivatives during the present study can successfully be employed for their large-scale production in bioreactors.