The availability of a system for direct transfer of antifungal candidate genes into European chestnut (Castanea sativa Mill.) would offer an alternative approach to conventional breeding for production of chestnut trees tolerant to ink disease caused by Phytophthora spp. For the first time, a chestnut thaumatin-like protein gene (CsTL1), isolated from chestnut cotyledons, has been overexpressed in three chestnut somatic embryogenic lines. Transformation experiments have been performed using an Agrobacterium tumefaciens Smith and Townsend vector harboring the neomycin phosphotransferase (NPTII) selectable and the green fluorescent protein (EGFP) reporter genes. The transformation efficiency, determined on the basis of the fluorescence of surviving explants, was clearly genotype dependent and ranged from 32.5% in the CI-9 line to 7.1% in the CI-3 line. A total of 126 independent transformed lines were obtained. The presence and integration of chestnut CsTL1 in genomic DNA was confirmed by polymerase chain reaction (PCR) and Southern blot analyses. Quantitative real-time PCR revealed that CsTL1 expression was up to 13.5-fold higher in a transgenic line compared with its corresponding untransformed line. In only one of the 11 transformed lines tested, expression of the CsTL1 was lower than the control. The remaining 115 transformed lines were successfully subjected to cryopreservation. Embryo proliferation was achieved in all of the transgenic lines regenerated and the transformed lines showed a higher mean number of cotyledonary stage embryos and total number of embryos per embryo clump than their corresponding untransformed lines. Transgenic plants were regenerated after maturation and germination of transformed somatic embryos. Furthermore, due to the low plantlet conversion achieved, axillary shoot proliferation cultures were established from partially germinated embryos (only shoot development), which were multiplied and rooted according to procedures already established. Transgenic plants were acclimatized and grown in a greenhouse. No phenotypic differences were found with control plants, suggesting no potential cytotoxic effects of the green fluorescent protein. The results reported in the present work could be considered as a first step toward the production of fungal-disease tolerant cisgenic chestnut plants.