Abstract CD45 is a receptor-like tyrosine phosphatase that positively regulates BCR signaling by dephosphorylating the inhibitory tyrosine of the Src family kinases. We showed previously that a single point mutation, E613R, introduced into the cytoplasmic membrane–proximal “wedge” domain of CD45 is sufficient to drive a lupus-like autoimmune disease on a susceptible genetic background. To clarify the molecular mechanism of this disease, we took advantage of a unique allelic series of mice in which the expression of CD45 is varied across a broad range. Although both E613R B cells and those with supraphysiologic CD45 expression exhibited hyperresponsive BCR signaling, they did so by opposite regulation of the Src family kinase Lyn. We demonstrated that the E613R allele of CD45 does not function as a hyper- or hypomorphic allele but rather alters the substrate specificity of CD45 for Lyn. Despite similarly enhancing BCR signaling, only B cells with supraphysiologic CD45 expression became anergic, whereas only mice harboring the E613R mutation developed frank autoimmunity on a susceptible genetic background. We showed that selective impairment of a Lyn-dependent negative-regulatory circuit in E613R B cells drove autoimmunity in E613R mice. This demonstrates that relaxing negative regulation of BCR signaling, rather than enhancing positive regulation, is critical for driving autoimmunity in this system.