CD200 and its receptor CD200R are type-1 membrane glycoproteins, which contain two immunoglobulin-like domains. Engagement of CD200R by CD200 inhibits activation of myeloid cells. Unlike the majority of immune inhibitory receptors, CD200R does not contain an immunoreceptor tyrosine-based inhibitory motif but contains three tyrosine residues (Y286, Y289, and Y297) in the cytoplasmic domain. Y297 is located in an NPxY motif. Previously, we have shown that engagement of CD200R in mouse mast cells induces its tyrosine phosphorylation and recruitment of inhibitory adaptor proteins Dok1 and Dok2, leading to the inhibition of Ras/mitogen-activated protein kinase activation. In the present study, we examined the roles of these three tyrosines in CD200R-mediated inhibition by site-directed mutagenesis in mouse mast cells. Our data show that Y286 and Y297 are the major phosphorylation sites and are critical for CD200R-mediated inhibition of mast cell activation, and Y289 is dispensable. Our data also suggest that the Src family kinase may mediate the phosphorylation of CD200R and Dok.