Background: In plant micropropagation, the adaptation phase represents one of the most critical limiting steps due to the high mortality rates commonly observed under ex-vitro conditions. The Fercal grapevine rootstock, characterized by its high adaptability to calcareous soils, holds significant potential for large-scale propagation. Therefore, evaluating strategies to improve survival during the transition from in vitro to ex-vitro environments is essential. This study investigates the influence of different growing media and foliar-applied chitosan doses on the adaptation success of Fercal rootstock plantlets. Results: After transferring the plantlets to media containing either cocopeat or black sand, foliar chitosan treatments were applied at concentrations of 0, 15, and 30 mg L−1. Morphological and physiological parameters such as survival rate (%), shoot length (cm), shoot diameter (mm), internode length (cm), leaf area (cm²), number of nodes (per plant), number of leaves (per plant), and chlorophyll content (SPAD value) were systematically evaluated. The results showed that chitosan treatments significantly improved survival compared to the control group, which exhibited complete mortality (0% survival) in both media. The highest survival rate, 66.66%, was obtained with 30 mg L−¹ chitosan in black sand, while the greatest shoot elongation (24.63 cm) was observed in cocopeat treated with 15 mg L−¹ chitosan. Morphological responses varied depending on both the chitosan concentration and the type of growing medium. Principal component analysis and hierarchical cluster analysis revealed strong correlations among internode length, shoot number, and survival rate. Conclusion: Chitosan is a promising bio-stimulant for enhancing the ex-vitro performance of grapevine rootstocks, with its effectiveness strongly influenced by the physicochemical characteristics of the growing substrate. The combined use of inert substrates with high chitosan doses or organic substrates with moderate doses resulted in optimal outcomes. These findings provide a basis for refining micropropagation protocols, especially under environmentally challenging conditions.