The four Ca(2+)-dependent NFATc proteins are both signal transducers and transcription factors that reside in the cytoplasm until dephosphorylation by calcineurin. Dephosphorylation exposes nuclear import sequences and sends NFATc proteins into the nucleus where they assemble with nuclear partners into NFAT transcription complexes. Recent genetic studies have indicated that calcineurin-NFAT signaling is a major determinant of vertebrate morphogenesis and development. Mice lacking calcineurin activity show a complete block in positive selection of CD4 and CD8 double-positive thymocytes, yet the role of the NFATc proteins in T cell development has been controversial. In this study, we address the requirement for NFATc3 in T cell development by generating NFATc3 conditional knockout mice. We show that specific deletion of NFATc3 in thymocytes causes a partial block at the double-negative stage 3 and also a partial block in positive selection. Furthermore, the defect does not become more pronounced when NFATc2 is also absent, consistent with the fact that NFATc2-null mice do not have a T cell developmental defect. Expression of a nuclear (and constitutively active) NFATc1 even at subphysiological levels can rescue the transition of double-negative to double-positive thymocytes in RAG-null mice, but is unable to rescue development of CD4 and CD8 single-positive cells. In addition to NFATc3, this suggests a role for NFATc1 in T cell development. Our studies indicate that the signals that direct positive selection likely use both NFATc1 and NFATc3 downstream of calcineurin.