miR-145 and miR-143 are co-transcribed in multipotent cardiac progenitors and are localized to smooth muscle cells, including neural crest stem cell-derived vascular smooth muscle cells (VSMCs). These microRNAs are direct transcriptional targets of serum response factor (SRF), myocardin, and Nkx2.5, and their expression is downregulated in injured or atherosclerotic vessels with proliferating, less differentiated VSMCs. miR-145 is essential for myocardin-induced reprogramming of adult fibroblasts into VSMCs and sufficient to induce differentiation of multipotent neural crest stem cells into vascular smooth muscle. miR-145 and miR-143 cooperatively target a network of transcription factors, including Klf4, myocardin, and Elk-1, to promote differentiation and repress proliferation of VSMCs. These findings demonstrate that miR-145 can direct the smooth muscle fate and that miR-145 and miR-143 function to regulate the quiescent versus proliferative phenotype of VSMCs.miR-145 and miR-143 are co-transcribed in multipotent cardiac progenitors and are localized to smooth muscle cells, including neural crest stem cell-derived vascular smooth muscle cells (VSMCs). These microRNAs are direct transcriptional targets of serum response factor (SRF), myocardin, and Nkx2.5, and their expression is downregulated in injured or atherosclerotic vessels with proliferating, less differentiated VSMCs. miR-145 is essential for myocardin-induced reprogramming of adult fibroblasts into VSMCs and sufficient to induce differentiation of multipotent neural crest stem cells into vascular smooth muscle. miR-145 and miR-143 cooperatively target a network of transcription factors, including Klf4, myocardin, and Elk-1, to promote differentiation and repress proliferation of VSMCs. These findings demonstrate that miR-145 can direct the smooth muscle fate and that miR-145 and miR-143 function to regulate the quiescent versus proliferative phenotype of VSMCs.