Thymic metallophilic macrophages represent a significant component in the thymus physiology. Recently, we showed their presence to be dependent on functional lymphotoxin-beta receptor (LT beta R) signaling pathway. However, it is unknown whether the development of metallophilic macrophages also requires the Autoimmune regulator (Aire) transcription factor, as suggested by some studies for medullary thymic epithelial cells, or perhaps the presence of Aire-expressing thymic epithelial cells themselves. Therefore, we investigated the presence of metallophilic macrophages in Aire-deficient thymus. Our study shows that the metallophilic macrophages are fully developed in the Aire-deficient thymus; their development is not regulated via Aire transcription factor and does not require the presence of Aire-expressing epithelial cells. On the contrary, in alymphoplasia (ALY) mice (deficient in nuclear factor-kappaB-inducing kinase, NIK), which we used as negative control, thymic metallophilic macrophages are completely lacking, similarly as in LT beta R-deficient animals. Together, these results show that the development/maintenance of thymic metallophilic macrophages is executed via LT beta R circumventing the Aire transcription factor. Thus, we shed a new light on the molecular requirements for development of these cells and also show that LT beta R pathway is a common developmental regulator of metallophilic macrophages in different lymphatic organs (i.e., thymus and spleen).