Cementum is formed continuously during the life of the tooth. The paravascular zones in the adult periodontal ligament (PL) comprise the progenitors of the fibroblastic (Fb), osteoblastic (Ob) and cementoblastic (Cb) lineages. A key question in studying cementogenesis is “What are the factors that control the differentiation of the Cb lineage?” The purpose of this study was to assess the effect of cementum attachment protein (CAP) on the differentiation of cloned PL progenitors. Cloned Cb, Ob and Fb progenitors were obtained from PL cultures; some of the clones were immortalized. The effect of CAP on the differentiation of cloned progenitors was tested by assessing the expression of alkaline phosphatase (AIP), CAP and bone sialoprotein (BSP) by immunochemistry (number of positive cells) and northern blots, and that of mineralized tissue by histochemistry. Untreated Fb clones were negative for all tested markers. The CAP expression in Cb untreated clones was 2-fold higher than that of Ob ones, whereas BSP expression was similar in both immunochemistry and northern blots. CAP changed the pattern of mineralized tissue formation in the Ob clones from nodular to a continuous ridge-like pattern characteristic to that formed by Cb clones and cementoma derived cell lines. CAP induced the expression of AIP, CAP, BSP and mineralized tissue formation in some of the Fb clones. These results indicate, for the first time, that CAP regulates the differentiation of the Cb lineage by inducing and enhancing the differentiation of putative cementoblastic progenitors and has the capacity to recruit fibroblastic progenitors to the cementoblastic lineage.