The aggregation of mostly antibody light chain variable (V_L) domains into amyloid fibrils in various tissues is the main cause of death in systemic amyloid light chain amyloidosis. Point mutations within the domain are important to shift the V_L into the fibrillar pathway, but why and how only some site-specific mutations achieve this still remains elusive. We show here that both destabilizing and surprisingly stable mutants readily predispose an amyloid-resistant V_L domain to amyloid formation. The decreased thermodynamic stability of the destabilizing mutant results in the accumulation of non-native intermediates that readily populate the amyloid state. Interestingly, the stable mutants establish site-specific non-native interactions with especially nearby serine/threonine residues that unexpectedly do not affect the folding behavior of the V_L domain but rather readily induce and stabilize the fibril structure, a previously unrecognized mechanism. These findings provide a new concept for the molecular mechanism of amyloid fibril formation.