Many protein-protein interaction domains bind to multiple targets. However, little is known about how the interactions of a single domain with many proteins are controlled and modulated under varying cellular conditions. In this study, we investigated the in vivo effects of Abp1p SH3 domain mutants that incrementally reduce target-binding affinity in four different yeast mutant backgrounds in which Abp1p activity is essential for growth. Although the severity of the phenotypic defects observed generally increased as binding affinity was reduced, some genetic backgrounds (prk1 Delta and sla1 Delta) tolerated large affinity reductions while others (sac6 Delta and sla2 Delta) were much more sensitive to these reductions. To elucidate the mechanisms behind these observations, we determined that Ark1p is the most important Abp1p SH3 domain interactor in prk1 Delta cells, but that interactions with multiple targets, including Ark1p and Scp1p, are required in the sac6 Delta background. We establish that the Abp1p SH3 domain makes different, functionally important interactions under different genetic conditions, and these changes in function are reflected by changes in the binding affinity requirement of the domain. These data provide the first evidence of biological relevance for any Abp1p SH3 domain-mediated interaction. We also find that considerable reductions in binding affinity are tolerated by the cell with little effect on growth rate, even when the actin cytoskeletal morphology is significantly perturbed.