Wood biomass is mainly made of secondary cell walls; hence, elucidation of the molecular mechanisms underlying the transcriptional regulation of secondary wall biosynthesis during wood formation will be instrumental to design strategies for genetic improvement of wood biomass. Here, we provide direct evidence demonstrating that the poplar (Populus trichocarpa) wood-associated NAC domain transcription factors (PtrWNDs) are master switches activating a suite of downstream transcription factors, and together, they are involved in the coordinated regulation of secondary wall biosynthesis during wood formation. We show that transgenic poplar plants with dominant repression of PtrWNDs functions exhibit a drastic reduction in secondary wall thickening in woody cells, and those with PtrWND overexpression result in ectopic deposition of secondary walls. Analysis of PtrWND2B overexpressors revealed up-regulation of the expression of a number of wood-associated transcription factors, the promoters of which were also activated by PtrWND6B and the Eucalyptus EgWND1. Transactivation analysis and electrophoretic mobility shift assay demonstrated that PtrWNDs and EgWND1 activated gene expression through direct binding to the secondary wall NAC-binding elements, which are present in the promoters of several wood-associated transcription factors and a number of genes involved in secondary wall biosynthesis and modification. The WND-regulated transcription factors PtrNAC150, PtrNAC156, PtrNAC157, PtrMYB18, PtrMYB74, PtrMYB75, PtrMYB121, PtrMYB128, PtrZF1, and PtrGATA8 were able to activate the promoter activities of the biosynthetic genes for all three major wood components. Our study has uncovered that the WND master switches together with a battery of their downstream transcription factors form a transcriptional network controlling secondary wall biosynthesis during wood formation.Wood, composed of cellulose, hemicelluloses, and lignin, is the most abundant biomass produced by plants. It is an important raw forest product that has traditionally been used for myriad applications, including construction, pulping, paper making, direct burning for energy, and so on. Recently, wood from tree species, such as poplar (Populus spp.), has been proposed to be a renewable source for biofuel production (Carroll and Somerville, 2009). Therefore, there is a surge of interest in elucidating the molecular mechanisms underlying the process of wood formation in the hope of developing strategies for increasing wood biomass production and/or modifying wood composition tailored for biofuel production. Since wood biomass is mainly made of secondary walls, elucidation of the mechanisms underlying the coordinated activation of secondary wall biosynthetic genes will undoubtedly contribute to our understanding of the molecular control of wood formation.Molecular and genomic studies in tree species have uncovered a number of wood-associated transcription factors that might be involved in the coordinated regulation of wood formation (Patzlaff et al