Animal development is an elaborate process programmed by genomic regulatory instructions. Regulatory genes encode transcription factors and signal molecules, and their expression is under the control of cis-regulatory modules that define the logic of transcriptional responses to the inputs of other regulatory genes. The functional linkages among regulatory genes constitute the gene regulatory networks (GRNs) that govern cell specification and patterning in development. Constructing such networks requires identification of the regulatory genes involved and characterization of their temporal and spatial expression patterns. Interactions (activation/repression) among transcription factors or signals can be investigated by large-scale perturbation analysis, in which the function of each gene is specifically blocked. Resultant expression changes are then integrated to identify direct linkages, and to reveal the structure of the GRN. Predicted GRN linkages can be tested and verified by cis-regulatory analysis. The explanatory power of the GRN was shown in the lineage specification of sea urchin endomesoderm. Acquiring such networks is essential for a systematic and mechanistic understanding of the developmental process. Birth Defects Research (Part C) 87:123–130, 2009. © 2009 Wiley-Liss, Inc.