The calcineurin/nuclear factor of activated T cells (NFAT) signaling pathway is best known for its role in T lymphocyte activation. However, it has become increasingly apparent that this signaling pathway is also involved in the regulation of cell growth and development in a wide variety of different tissues and cell types. Here we have investigated the effects of sustained NFATc1 signaling on the growth and differentiation of the murine 3T3-L1 preadipocyte cell line. Remarkably, we find that expression of a constitutively active NFATc1 mutant (caNFATc1) in these immortalized cells inhibits their differentiation into mature adipocytes and causes them to adopt a transformed cell phenotype, including loss of contact-mediated growth inhibition, reduced serum growth requirements, protection from growth factor withdrawal-induced apoptosis, and formation of colonies in semisolid media. Furthermore, we find that caNFATc1-expressing cells acquire growth factor autonomy and are able to proliferate even in the complete absence of serum. We provide evidence that this growth factor independence is caused by the NFATc1-dependent production of a soluble heat-labile autocrine factor that is capable of promoting the growth and survival of wild type 3T3-L1 cells as well as potently inhibiting their differentiation into mature adipocytes. Finally, we demonstrate that cells expressing caNFATc1 form tumors in nude mice. Taken together, these results indicate that deregulated NFATc1 activity is able to induce the immortalized 3T3-L1 preadipocyte cell line to acquire the well established hallmarks of cellular transformation and thereby provide direct evidence for the oncogenic potential of the NFATc1 transcription factor.