Central to the normal function of the immune system is its ability to distinguish between self and non-self since failure to do so could provoke the onset of autoimmune disease. To avoid this possibility, the immune system employs several processes that include, negative selection, peripheral tolerance, and limiting DC antigen priming of naïve T cells to the lymph nodes. Naïve T cells must receive two independent signals from these antigen-presenting cells (APC) that other cells cannot provide if they are to become productively activated. The first is antigen-specific and occurs when T cell antigen receptors encounter the appropriate antigen-MHC complex on the APC--Signal 1. A second, antigen-independent signal is delivered through a T cell costimulatory molecule that engages its APC-expressed ligands--Signal 2. In the absence of a costimulatory signal T cells typically enter a state of anergy. Furthermore, the extent to which T cell activation occurs can be held in check through specific inhibitory receptors expressed on T cells. Understanding the basic mechanisms of how T cell activation is regulated has led to the development of therapeutic approaches for targeting T cell costimulatory and inhibitory pathways for turning on, or preventing the turning off immune responses in subjects with cancer. In this review we will discuss several T cell costimulatory and inhibitory pathways known to influence the development of anti-tumor immunity and how experimental manipulation of these signaling pathways has led to the generation of protective, or curative anti-tumor immunity in mice and humans.