TBX3, the gene mutated in ulnar-mammary syndrome (UMS), is involved in the production of a transcription factor of the T-box family, known to inhibit transcription from the p14ARF (p19ARF in mouse) promoter in fibroblasts and to contribute to cell immortalization. One of the main features of the UMS phenotype is the severe hypoplasia of the breast, associated with haploinsufficiency of the TBX3 gene product. In mice homozygous for the targeted disruption of Tbx3, the mammary glands (MGs) are nearly absent from early stages of embryogenesis, whereas in heterozygous adults, the MGs show reduced ductal branching. All these data strongly suggest a specific role of TBX3 in promoting the growth of mammary epithelial cells (MECs), although direct evidence of this is lacking. Here, we provide data showing the growth-promoting function of Tbx3 in several models of MECs, in association with its ability to repress the ARF promoter. However, no effect of Tbx3 on cell differentiation or apoptosis has been observed. The growth promoting function also entails the down-regulation of p21 CIP1/WAF and an increase in cyclin D1 but is independent of p53 and Mdm2 cell-cycle regulatory proteins, as p53-null MECs show similar growth responses associated with the up- or down-regulation of Tbx3. This is the first direct evidence that the level of Tbx3 expression positively controls the proliferation of MECs via pathways alternative to Mdm2-p53.