Arabidopsis root development provides a remarkably tractable system to delineate tissue-specific, developmental gene regulatory networks and to study their functionality in a complex multicellular model system over developmental time. Tightly controlled gene expression within tissues is a hallmark of multicellular development and is accomplished by transcription factors (TFs) and microRNAs (miRNAs). We present an automated, enhanced yeast one hybrid (eY1H) assay using a tissue-specific TF resource to comprehensively map gene regulatory networks in the Arabidopsis root stele. These gene regulatory networks are robust and highly combinatorial in nature. Using these methods and computational modeling, we have additionally modeled a gene regulatory network that regulates distinct transcriptional events in developmental time. Distinct regulatory modules were identified that temporally drive the expression of genes involved in xylem specification and in the subsequent synthesis of secondary cell wall metabolites associated with xylem differentiation.