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Abstract: Mammalian color patterns are extremely diverse and can have a profound impact on fitness. However, little is known about the mechanisms underlying their formation and evolution. I took advantage of the naturally occurring color pattern of the African striped mice, Rhabdomys pumilio, to investigate the processes responsible for forming periodic stripes, a common pattern in mammals. De novo transcriptomic assembly and analysis of striped mouse skin showed that stripes result from underlying differences in melanocyte maturation, which give rise to spatial variation in hair color. From the many loci involved in this process, the transcription factor Alx3 was identified as a major hierarchical regulator. During striped mouse embryogenesis, patterned expression of Alx3 precedes pigment stripes and acts to directly represses Mitf, a master regulator of melanocyte differentiation. In addition, Alx3 is also differentially expressed in the dorsal stripes of chipmunks, which have independently evolved a similar pattern of dorsal stripes. Together, this work shows that differences in the spatial control of Alx3 expression lead to striped patterns in rodents, revealing both a new factor regulating pigment cells and a previously unappreciated mechanism for modulating hair color, and thereby provide new insight into the ways in which phenotypic novelty evolves.