Members of the basic helix-loop-helix (bHLH) gene family play important roles in vertebrate neurogenesis. In this study, confocal microscopy-based fluorescence resonance energy transfer (FRET) is used to monitor bHLH protein-protein interactions under various physiological conditions. Tissue-specific bHLH activators, NeuroD1, Mash1, Neurogenin1 (Ngn1), Neurogenin2 (Ngn2), and ubiquitous expressed E47 protein are tagged with enhanced yellow fluorescence protein (EYFP) and enhanced cyan fluorescence protein (ECFP), respectively. The subcellular localization and mobility of bHLH fusion proteins are examined in HEK293 cells. By transient transfection and in ovo electroporation, four pairs of tissue-specific bHLH activators and E47 protein are over-expressed in HEK293 cells and developing chick embryo neural tube. With the acceptor photobleaching method, FRET could be detected between these bHLH protein pairs in the nuclei of transfected cells and developing neural tubes. Mash1/E47 and Ngn2/E47 FRET pairs show higher FRET efficiencies in the medial and the lateral half of chick embryo neural tube, respectively. It suggests that these bHLH protein pairs formed functional DNA-protein complexes with regulatory elements of their downstream target genes in the specific regions. This work will help one understand the behaviours of bHLH factors in vivo.