RNA polymerase (pol) III transcription, responsible for the synthesis of various stable RNAs, including 5S rRNAs and tRNAs, is regulated by oncogenic proteins and tumor suppressors. While it is well established that RNA pol III-dependent transcription is deregulated in transformed cells and malignant tumors, it has not been determined whether this represents a cause or consequence of these processes. We show that rat1a fibroblasts undergoing oncogenic transformation by the TATA-binding protein or c-myc display enhanced RNA pol III transcription. Decreased expression of the RNA pol III-specific transcription factor, Brf1, prevents this increase in RNA pol III transcription. While the overall proliferation rates of these cells remain unchanged, the ability of cells to grow in an anchorage-independent manner and form tumors in mice was markedly reduced. While overexpression of Brf1 modestly stimulated RNA pol III transcription, expression of a phospho-mimic, Brf1-T145D, more significantly induced transcription. However, these increases in transcription were not sufficient to promote cellular transformation. Together, these results demonstrate that enhanced RNA pol III transcription is essential for anchorage-independent growth and tumorigenesis, and that these events can be uncoupled from effects on anchorage-dependent proliferation.