Medicago truncatula is a forage crop that has been developed into a model legume. As a model plant, M. truncatula is particularly useful for the study of root endosymbiotic associations, including nodulation and mycorrhizal colonization. The development of different transformation methods is an important aspect for functional genomic studies in the species. This protocol describes an efficient system for generating transgenic plants from M. truncatula roots based on Agrobacterium tumefaciens-mediated transformation. Furthermore, the protocol can be easily adapted for recovering fertile transgenic plants from transformed hairy roots.