Mammalian color patterns are among the most recognizable characters found in nature and can have a profound impact on fitness. However, little is known about the mechanisms underlying their formation and subsequent evolution. Here we show that, in the African striped mouse (Rhabdomys pumilio), periodic dorsal stripes result from underlying differences in melanocyte maturation, which give rise to spatial variation in hair color, and we identify the transcription factor Alx3 as a regulator of this process. In embryonic dorsal skin, patterned expression of Alx3 foreshadows pigment stripes, and acts to directly repress Mitf, a master regulator of melanocyte differentiation, giving rise to light-colored hair. Moreover, Alx3 is also upregulated in the light stripes of chipmunks, which have independently evolved a similar pattern of dorsal stripes. Our results reveal a previously unappreciated mechanism for modulating spatial variation in hair color, and provide new insight into the ways in which phenotypic novelty evolves.