The deleterious side effects associated with a recent clinical trial with anti-CD28 superagonist Abs have questioned the use of reagents to costimulatory molecules in human therapy. We now show that sustained signaling from an agonist Ab to 4-1BB, a member of the TNFR superfamily, results in detrimental effects on immune cell homeostasis. Repeated anti-4-1BB treatment during the reconstitution of hematopoietic cells in irradiated mice engrafted with bone marrow, or in mice infected with vaccinia virus, induced abnormal apoptosis of premature and immature B cells in the bone marrow, and led to peripheral B cell depletion. Inhibition of B cell development was indirect and due to costimulation of CD8 T cells and dependent on IFN-gamma. Moreover, anti-4-1BB also suppressed the development of NK and NKT cells, but in this case independently of T cells and IFN-gamma. The altered NK cell homeostasis resulted from activation-induced cell death triggered by anti-4-1BB. These results show that hypercostimulation elicits strong T cell immunity, but it can simultaneously distort immune homeostasis, suggesting that careful attention to activity, dose, and periodicity of treatment will be needed in any immunotherapeutic strategy with agonist Abs to costimulatory molecules.