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The strength of Ca²⁺ signaling is a hallmark of T cell activation, yet the role of Ca²⁺ homeostasis in developing T cells before expressing a mature T cell receptor is poorly understood. We aimed to unveil specific functions of the two plasma membrane Ca²⁺ ATPases expressed in T cells, PMCA1 and PMCA4. On a transcriptional and protein level we found that PMCA4 was expressed at low levels in CD4⁻CD8⁻ double negative (DN) thymocytes and was even downregulated in subsequent stages while PMCA1 was present throughout development and upregulated in CD4⁺CD8⁺ double positive (DP) thymocytes. Mice with a targeted deletion of <i>Pmca1</i> in DN3 thymocytes had an almost complete block of DP thymocyte development with an accumulation of DN4 thymocytes but severely reduced numbers of CD8⁺ immature single positive (ISP) thymocytes. The DN4 thymocytes of these mice showed strongly elevated basal cytosolic Ca²⁺ levels and a pre-mature CD5 expression, but in contrast to the DP thymocytes they were only mildly prone to apoptosis. Surprisingly, mice with a germline deletion of <i>Pmca4</i> did not show any signs of altered progression through the developmental thymocyte stages, nor altered Ca²⁺ homeostasis throughout this process. PMCA1 is, therefore, non-redundant in keeping cellular Ca²⁺ levels low in the early thymocyte development required for the DN to DP transition.