The Arabidopsis BYPASS1 (BPS1) gene is required for normal root and shoot development. In bps1 mutants, grafting and root excision experiments have shown that mutant roots produce a transmissible signal that is capable of arresting shoot development. In addition, we previously showed that growth of bps1 mutants on the carotenoid biosynthesis inhibitor fluridone resulted in partial rescue of both leaf and root defects. These observations suggest that a single mobile carotenoid-derived signal affects both root and shoot development. Here, we describe further characterization of the bps1 root-derived signal using genetic and biosynthetic inhibitor approaches. We characterized leaf and root development in double mutants that combined the bps1 mutant with mutants that have known defects in genes encoding carotenoid processing enzymes or defects in responses to carotenoid-derived abscisic acid. Our studies indicate that the mobile signal is neither abscisic acid nor the MAX-dependent hormone that regulates shoot branching, and that production of the signal does not require the activity of any single carotenoid cleavage dioxygenase. In addition, our studies with CPTA, a lycopene cyclase inhibitor, show that signal production requires synthesis of β-carotene and its derivatives. Furthermore, we show a direct requirement for carotenoids as signal precursors, as the GUN plastid-to-nucleus signaling pathway is not required for phenotypic rescue. Together, our results suggest that bps1 roots produce a novel mobile carotenoid-derived signaling compound.