Timothy (Phleum pratense) is a widely grown perennial forage grass in the Nordic region. The canopy consists of three tiller types, of which the stem forming vegetative elongating (ELONG) tiller and generative (GEN) tillers contribute the most to dry matter yield. In this study, the regulation of tiller formation by vernalization, day length (DL) [12 h, short day length (SD); 16 h, long day length (LD)] and gibberellic acid (GA) was investigated in two timothy cultivars. Vernalization resulted in a shift of ELONG to GEN tillers. No vernalization was required for the development of ELONG tillers but SD strictly arrested stem elongation. Vernalization is an important regulator of tiller development but it seemed to be upstream regulated by DL. LD was essential for floral transition and could not be substituted by GA and/or vernalization treatments. Genotypic variation was found in the development of GEN tillers. The ability to produce GEN tillers was associated with significant upregulation of PpVRN3. PpVRN1 expression peaked at the time of vegetative/generative transition, and PpVRN3 after the transfer to LD, suggesting them to have similar functions with cereal vernalization genes. PpVRN1 alone was not sufficient to activate flowering, and upregulation of PpVRN3 possibly together with PpPpd1 was required. Although vernalization downregulated PpMADS10, this gene did not act as a clear flowering repressor. Our results show that flowering signals alter the tiller composition, so they have important effects on yield formation of timothy.