ZAP-70 is critical for T cell receptor (TCR) signaling. Tyrosine to phenylalanine mutations of Y315 and Y319 in ZAP-70 suggest these residues function to recruit downstream effector molecules, but mutagenesis and crystallization studies reveal that these residues also play an important role in autoinhibition ZAP-70. To address the importance of the scaffolding function, we generated a zap70 mutant mouse (YYAA mouse) with Y315 and Y319 both mutated to alanines. These YYAA mice reveal that the scaffolding function is important for normal development and function. Moreover, the YYAA mice have many similarities to a previously identified ZAP-70 mutant mouse, SKG, which harbors a distinct hypomorphic mutation. Both YYAA and SKG mice have impaired T cell development and hyporesponsiveness to TCR stimulation, markedly reduced numbers of thymic T regulatory cells and defective positive and negative selection. YYAA mice, like SKG mice, develop rheumatoid factor antibodies, but fail to develop autoimmune arthritis. Signaling differences that result from ZAP-70 mutations appear to skew the TCR repertoire in ways that differentially influence propensity to autoimmunity versus autoimmune disease susceptibility. By uncoupling the relative contribution from T regulatory cells and TCR repertoire during thymic selection, our data help to identify events that may be important, but alone are insufficient, for the development of autoimmune disease.