Transcription factors regulate gene expression by directly binding the cis-acting regulatory elements of target genes via their DNA-binding domains or by interacting with other transcription factors. Trichome cell fate determination in Arabidopsis utilizes a lateral inhibition mechanism that relies on the interplay of transcription factors. GLABRA1 (GL1), an R2R3 MYB transcription factor, GLABRA3 (GL3), a basic helix-loop-helix (bHLH) transcription factor, and TRANSPARENT TESTA GLABRA1 (TTG1), a WD40 protein, are believed to form a transcriptional activator complex to control the transcription of GLABRA2 (GL2), which in turn induces trichome formation in shoots. However, the molecular mechanism of the regulation of GL2 expression by this activator complex is still poorly understood. Here we report that GL1 and GL3 control GL2 expression by a previously unrecognized mechanism in which in addition to the protein-protein interaction between GL1 and GL3, concurrent binding of GL1 and GL3 to the promoter of GL2 via their own DNA-binding domains is probably required to activate GL2. We demonstrate that disruption or deletion of the DNA-binding domains in either GL1 or GL3 completely abolishes the transcriptional activity of the GL1-GL3 complex in activating GL2. These results provide new insight into the interplay of GL1 and GL3 transcription factors in the activation of GL2.