The p16-CyclinD1-CDK4-RB pathway regulates G1-S cell cycle transition. This pathway is deregulated in 90% of melanomas, through amplification or increased expression of CDK4 or Cyclin D1 (CCND1), or reduced expression of the CDK inhibitor p16INK4A (CDKN2A). To explore if CDK4 may be a viable target for the treatment of human melanoma, we screened a panel of melanoma cell lines with the highly selective CDK4/6 inhibitor PD-0332991 (PD-991). Expression and mutational analysis was performed to identify additional genomic predictors of sensitivity and resistance. A majority of cell lines were found to be sensitive to PD-991, although there was a distinct population of resistant cell lines. Loss of CDKN2A/p16 correlated with sensitivity to PD-991 (p<0.02). Mutation of p53 and the corresponding low expression of p53 targets p21 and MDM2 correlated with resistance (p<0.0001). In sensitive melanoma cell lines, treatment with PD-991 resulted in loss of CDK4 targets FOXM1 and hyperphosphorylated RB1 and a concomitant G0/G1 cell cycle arrest. Treated cells displayed an enlarged flattened morphology and an increase in SA-β-galactosidase staining compared to vehicle controls, indicative of cellular senescence. RB1-null cell lines were resistant to CDK4/6 inhibition, and did not display cell cycle arrest or senescence. An acquired resistance (AR) cell line was derived by growing a sensitive cell line under continuous drug pressure. AR cells showed a partial cell cycle arrest in response to PD991, but were able to proliferate better under drug pressure than the parental line. Western blots indicate that AR cells partially retain phosphorylated RB1 after drug treatment, suggesting potential reactivation of this pathway. These data support further evaluation of CDK4/6 inhibitors in melanoma, and suggest potential mechanisms for overcoming resistance.