Physiological stress resulting from infections, trauma, surgery, alcoholism, malnutrition, and/or pregnancy results in a substantial depletion of immature CD4(+)CD8(+) thymocytes. We previously identified 18 distinct stress-responsive microRNAs (miRs) in the thymus upon systemic stress induced by lipopolysaccharide (LPS) or the synthetic glucocorticoid, dexamethasone (Dex). MiRs are short, non-coding RNAs that play critical roles in the immune system by targeting diverse mRNAs, suggesting that their modulation in the thymus in response to stress could impact thymopoiesis. MiR-181d is one such stress-responsive miR, exhibiting a 15-fold down-regulation in expression. We utilized both transgenic and gene-targeting approaches to study the impact of miR-181d on thymopoiesis under normal and stress conditions. The over-expression of miR-181d in developing thymocytes reduced the total number of immature CD4(+)CD8(+) thymocytes. LPS or Dex injections caused a 4-fold greater loss of these cells when compared with the wild type controls. A knockout mouse was developed to selectively eliminate miR-181d, leaving the closely spaced and contiguous family member miR-181c intact. The targeted elimination of just miR-181d resulted in a thymus stress-responsiveness similar to wild-type mice. These experiments suggest that one or more of three other miR-181 family members have overlapping or compensatory functions. Gene expression comparisons of thymocytes from the wild type versus transgenic mice indicated that miR-181d targets a number of stress, metabolic, and signaling pathways. These findings demonstrate that selected miRs enhance stress-mediated thymic involution in vivo.