Plants have developed complex molecular signaling cascades to cope with fluctuations in weather conditions, effectively managing the delicate equilibrium between growth and survival. Our results underscore the interaction between light receptor phytochrome A (phyA) and histidine-containing phosphotransfer protein AHP3, the essential component of multistep phosphorelay (MSP). Employing FLIM-FRET, we elucidated the functional consequences of a mutation in the serine box of AHP3, previously identified as a phyA target site. Our analysis involved tracking the dynamic protein-protein interactions in different cellular compartments, capturing the translocation of both phyA and AHP3 proteins from the cytosol to the nucleus upon activation. The semi-quantitative output of FLIM-FRET proved invaluable, enabling statistical comparisons of changes in donor lifetime based on the presence of putative interactors. Notably, the phosphomimetic AHP3 mutant exhibited reduced affinity to histidine kinase receptors, while the phospho-deficient AHP3 mutant maintained the interaction affinity comparable to the wild-type AHP3. This finding highlighted the significance of phyA-dependent phosphorylation in modulating the transfer of phosphate moieties between AHP3 and cytokinin receptors, a crucial aspect of the cytokinin signaling-mediating MSP pathway. Verification of our observations using genetic tools further demonstrated the early negative impact of phyA on the MSP, leading to a transient reduction in the cytokinin signaling activity. Thus, FLIM-FRET analysis provides valuable insights into the dynamic regulatory networks integrating signaling pathways to govern the adaptive responses of plants.