As thymocytes undergo differentiation in the thymus, they progress through distinct phases of quiescence and proliferation. Identifying cellular mechanisms that maintain thymocytes in a non-dividing state is critical to fully understanding thymocyte development. Recently, TOB1 was identified as a key mediator of the quiescent state in peripheral anergic and unstimulated T cells. We demonstrate by RT-PCR, that of the five members of the BTG/TOB family present within the mouse genome, BTG3, TIS21 and TOB2 are expressed at significant levels in thymocytes. Further analysis revealed that their expression is decreased in the DN2 and beta-selected populations where proliferation is induced; suggesting that expression of some BTG/TOB family members is required for thymocytes to remain quiescent in the absence of mitogenic signals. Overexpression of TIS21 and TOB2, with a retroviral vector, in FTOC and OP9-DL1 co-culture demonstrated that these members can prevent the expansion of thymocytes at key stages of thymocyte development.