AbstractAs 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 understand T cell development. A member of the B cell translocation gene/transducer of ErbB‐2 (BTG/TOB) family of anti‐proliferative proteins was identified as a key mediator of the quiescent state in peripheral anergic and unstimulated T cells. Here, we demonstrate that the BTG/TOB family member TPA‐inducible sequence 21 (TIS21) is expressed in quiescent CD44+CD25– early progenitor thymocytes and CD44–CD25+ cells prior to TCR β‐selection. However, TIS21 expression is decreased in proliferating CD25+CD44+ progenitor thymocytes and CD25lowCD44– β‐selected cells, suggesting that its regulated expression may enable thymocytes to remain quiescent in the absence of mitogenic signals. We addressed the role of TIS21 in regulating thymocyte stage‐specific expansion by ectopically expressing TIS21 in developing thymocytes and hematopoietic progenitors. Dysregulated expression of TIS21 inhibited the expansion of thymocytes even in the presence of endogenous mitogenic signals, while thymocyte differentiation was unimpeded. These findings imply that the intracellular mechanisms regulating thymocyte differentiation and proliferation, which are induced downstream of developmental cues, function independently during early T cell development.