Antigen-mediated cross-linking of IgE bound to its receptor, FcRI, stimulates degranulation, phospholipid metabolism, and cytokine production in mast cells and basophils to initiate inflammatory and allergic responses. Previous studies suggested that spatial organization of the clustered receptors affects the assembly of the transmembrane signaling complexes. To investigate systematically the structural constraints in signal initiation, we utilized rigid double-stranded DNA scaffolds to synthesize ligands with tunable lengths. We characterized a series of symmetric trivalent DNA ligands with rigid spacing between 2,4-dinitrophenyl (DNP) haptenic groups in the range of 5-15 nm. These ligands all bind to anti-DNP IgE on RBL mast cells with similar avidity, and they all cross-link IgE-FcRI complexes effectively. We observe length-dependent stimulation of tyrosine phosphorylation of FcRI beta and gamma subunits and the adaptor protein LAT: the shortest ligand is approximately 5-10-fold more potent than the longest. Stimulated Ca2+ mobilization and degranulation also exhibits kinetics and magnitudes that differ as a function of ligand length. In contrast, tyrosine phosphorylation of phospholipase Cgamma1 and consequent Ca2+ release from intracellular stores do not show this dependence on ligand length. Our results with these rigid, DNA-based ligands provide direct support for receptor transphosphorylation as a key step in amplified signaling leading to degranulation, and they further reveal branching of pathways in signaling events.