Recent studies suggest that glial cells actively participate in the formation, function, maintenance, and repair of the chemical synapse. However, the molecular mechanisms of glia-synapse interactions are largely unknown. We have shown previously that Schwann cell-conditioned medium (SC-CM) promotes synaptogenesis in Xenopus nerve-muscle cocultures. The present study aimed to identify the synaptogenic molecules in SC-CM. Combining biochemical approaches and in vitro bioassays, we found that SC-CM contains transforming growth factor (TGF)-beta1, which is expressed in Schwann cells both in vivo and in vitro. Similar to SC-CM, TGF-beta1 doubled the size of acetylcholine receptor (AChR) clusters at nerve-muscle contacts and significantly increased the percentage of nerve-muscle contacts that show AChR clusters to approximately 60%, compared with approximately 20% seen in control cultures. The synaptogenic effects of SC-CM were abolished if SC-CM was immunodepleted of TGF-beta1 or if the latency-associated protein or a TGF-beta1 receptor kinase inhibitor was added to block the bioactivity of TGF-beta1. Similar to frog SC-CM, mammalian SC-CM also showed synaptogenic effects, which were prevented by immunodepletion of TGF-beta1. TGF-beta1 upregulated agrin expression in spinal neurons, which could explain the increase in AChR clusters in cultures treated with SC-CM. These results suggest that Schwann cells express TGF-beta1, which is both sufficient and necessary for mediating the synapse-promoting effects of Schwann cells at the developing neuromuscular junction. Schwann cell-derived TGF-beta1 thus joins other astrocyte-derived synaptogenic factors in further strengthening the emerging concept that glial cells contribute to synaptogenesis in both the PNS and the CNS.