The interplay between different signaling pathways that occurs during the development of a root system is investigated by considering, by way of example, the role of effectors of the Ca2+ signaling system and a rhizogenesis-stimulating oligosaccharin (OSRG) on the indoleacetic acid (IAA)-induced formation of adventitious roots. By identifying the sensitivity of different stages of rhizogenesis in cultivated buckwheat hypocotyls (Fagopyrum esculentum Moench) to IAA, Ca2+, and the oligosaccharin, it is shown that both Ca2+ and OSRG affect the root formation processes at its initial stages, preceding the effect of the hormone. Auxin can be added to the medium with some delay but no later than 1 h after the explant cultivation was started; while Ca2+ must be present in the medium from the very beginning of cultivation process. The number of IAA-induced adventitious roots on segments of buckwheat hypocotyls decreases in the presence of Ca2+ channel blockers (verapamil and diltiazem), compounds affecting Ca2+-release from intracellular storages (ruthenium red, neomycin), and calmodulin-antagonists (chlorpromazine and fluphenazine). Using calcium channel blocker (diltiazem), it is shown that the effect caused by the oligosaccharin does not depend on the functioning of potential-dependent channels of plasma membrane. The analysis of transcriptome profiling data for the cultivated explants identifies the expression of 149 genes (TGR > 5) relevant to Ca2+ signaling in investigated specimens. The expression profile of these genes changes markedly during IAA-induced root formation, while the addition of OSRG affecting the expression of only few of those.