Signal transduction across the plasma membrane via class I/II cytokine receptors requires assembly of complexes from two or more receptor subunits, which are non-covalently associated with Janus family tyrosine kinases (JAKs). Stoichiometry and composition as well as conformational organization and dynamics of such signaling complexes are determined by subtle, cooperative interactions. We aim to disentangle the determinants governing complex assembly in the cellular context in a quantitative manner by dedicated multicolor single-molecule imaging techniques. Leveraging highly efficient, cell surface-selective fluorescence labeling in conjunction with the enhanced capabilities of a newly designed TIRF-SIM microscope, we implemented single-molecule FRET (smFRET) to quantify assembly and conformational organization of receptor dimers in the plasma membrane of live cells. While we could rule out pre-assembly of receptor subunits in the absence of agonists, we uncovered that that diverse oncogenic receptor and JAK mutations initiate constitutive dimerization. Detailed conformational analysis by smFRET in combination with molecular dynamics simulations suggest that the formation of active signaling complexes critically depends on partially cooperative weak protein-protein and protein-lipid interactions encoded in the transmembrane and cytosolic receptor domains. Based on these insights, we propose a novel concept to rationalize the structural and functional plasticity of cytokine receptor signaling complexes.