Spleen tyrosine kinase (Syk) is a nonreceptor tyrosine kinase that is expressed primarily in hematopoietic cells. Because this protein has been implicated in processes such as Fc-mediated phagocytosis, BCR signaling, oxidative burst, degranulation, cytokine secretion, and integrin-mediated outside-in signaling, it is hypothesized that Syk may be a viable target in the treatment of a variety of autoimmune and inflammatory diseases. Because efforts to design a small-molecule therapeutic that specifically inhibits Syk have been largely unsuccessful, and genetic studies of Syk have been hampered by the fact that syk-/- mice die in utero, we have taken a chemical genetic approach to study the function of Syk. Specifically, we have created a mutant form of Syk that retains its wild-type function, but is susceptible to inhibition by enlarged derivatives of the tyrosine kinase inhibitor, PP1. We report in this study that Syk M442A S505A reconstituted wild-type function when introduced into murine syk-/- bone marrow-derived macrophages and syk-/- DT40 chicken B cells, as determined by functional and biochemical assays. Furthermore, after screening a series of PP1 derivatives, we identified one compound, namely 2,3-DMB-PP1, that specifically inhibited Syk M442A S505A, but not wild-type Syk. This system provides us with the power to characterize immune functions that are Syk specific, and furthermore, it provides us with a tool to assess how inhibition of Syk may alter an immune response and influence disease pathogenesis and/or progression.