p21 plays a dual role in keratinocyte growth and differentiation control. It restricts the number of keratinocyte stem cell populations while inhibiting the later stages of differentiation independently of the cell cycle. The molecular/biochemical mechanism for the differentiation suppressive function of p21 is unknown. Here we show that elevated p21 expression leads to activation of MAPK family members in a keratinocyte-specific and cell cycle-independent manner, and up-regulation of MAPK activity can explain the inhibitory effects of p21 on differentiation. p21 induces transcription of several genes with MAPK activation potential. Although several of these genes are induced by p21 in a MAPK-dependent manner, expression of IGF-I is induced upstream of MAPK activation. IGF-I stimulation is by itself sufficient to cause MAPK activation and inhibit differentiation and suppression of IGF-I signaling by knock down of the cognate receptor (IGF-R1), diminishing the ability of p21 to activate MAPK and suppress differentiation. Thus, in keratinocytes, the ability of p21 to suppress differentiation can be explained by cell type-specific activation of the MAPK cascade by transcriptional up-regulation of the IGF-I gene. p21WAF1/Cip1 was originally identified as a mediator of p53-induced growth arrest (1), a direct inhibitor of CDK 2 activity (CKI) (2), and a gene whose expression is induced with cellular senescence (3). This protein belongs to the Cip/Kip family of, p27Kip1 , and p57 Kip2), which share significant sequence homology in their amino-terminal portions and recognize a broad but not identical range of cyclin/CDK targets (4). The amino-terminal domain of p21, like the corresponding domains of p27 or p57, is both necessary and sufficient to inhibit cyclin/CDK activity in vitro and in vivo. The unique carboxyl-terminal domain of p21 associates with the proliferating nuclear antigen, a subunit of DNA polymerase ␦, and can inhibit DNA replication directly without affecting DNA repair (4). p21 has also been variously implicated as either a negative or positive regulator of apoptosis with cell type-specific mechanisms that may depend on its effects on the cell cycle or more direct association and modulation of proapoptotic molecules (5-7). Additionally p21 has been implicated in control of transcription through a mechanism that may be coupled to its CKI activity but also direct association and modulation of transcription factors and/or transcription coactivators (8 -11).This complexity of p21 function is well illustrated by its role in keratinocyte self-renewal and differentiation: p21 expression promotes the initial commitment of keratinocyte stem cell populations to differentiation (12) and contributes to differentiation-associated growth arrest (13). However, after an initial increase, at later stages of differentiation the p21 protein is down-modulated both in vitro and in the intact skin in vivo; sustained p21 expression under these conditions suppresses differentiation, suggesting that p21 is part of a negative r...