The transforming growth factor beta (TGF-β) pathway plays key roles in development and cancer. (TGF-β) signaling converges on the Smad2 and Smad3 effectors, which can either cooperate or antagonize to regulate their transcriptional targets. Here we performed in vivo and in silico experiments to study how such cooperativity and antagonism might function during neurogenesis. In vivo electroporation experiments in the chick embryo neural tube show that Smad2 and Smad3 cooperate to promote neurogenesis, as well as the transcription of Smad3 specific targets. Smad2 knockdown enhances neurogenesis and the transcription of Smad3 specific targets. A mathematical model of the TGF-β pathway fits the experimental results and predicts that the proportions of the three different trimeric complexes formed dictates the transcriptional responses of the R-Smads. As such, Smad2 targets are activated solely by the Smad2-Smad2-Smad4 complex, while Smad3 targets are activated both by Smad2-Smad3 Smad4 and Smad3- Smad3-Smad4 trimers. Since we have modeled the Smad responses onto arbitrary genes, we propose that this mechanism might be extended to additional activities of TGF-β in development and disease.