The cell surface molecule 2B4 (CD244) is an important regulator of lymphocyte activation, and its role in antiviral immunity and lymphoproliferative disorders is well established. Although it is also expressed on mast cells (MCs) and eosinophils (Eos), the functions of 2B4 on these allergy-orchestrating cells remain unclear. We therefore investigated the role of 2B4 on murine MCs and Eos, particularly how this molecule affects allergic and nonallergic inflammatory processes involving these effector cells. Experiments in bone marrow-derived cultures revealed an inhibitory effect for 2B4 in MC degranulation, but also an opposing stimulatory effect in eosinophil migration and delayed activation. Murine disease models supported the dual 2B4 function: In 2B4-/- mice with nonallergic peritonitis and mild atopic dermatitis (AD), modest infiltrates of Eos into the peritoneum and skin (respectively) confirmed that 2B4 boosts eosinophil trafficking. In a chronic AD model, 2B4-/- mice showed overdegranulated MCs, confirming the inhibiting 2B4 effect on MC activation. This multifunctional 2B4 profile unfolded in inflammation resembles a similar mixed effect of 2B4 in natural killer cells. Taken together, our findings provide evidence for physiological 2B4 stimulatory/inhibitory effects in MCs and Eos, pointing to a complex role for 2B4 in allergy.