Chikungunya virus (CHIKV) is an emerging arthropod-borne virus that has spread globally during the last two decades. The virus is mainly transmitted by Aedes aegypti and Aedes albopictus mosquitos and is thus capable of replicating in both human and mosquito cells. CHIKV has a broad tropism in vivo, capable of replicating in various tissues and cell types but largely excluding blood cells. This was reflected in vitro by a broad array of adherent cell lines supporting CHIKV infection. One marked exception to this general rule is the resistance of the lung cancer-derived A549 cell line to CHIKV infection. We verified that A549 cells were restrictive to infection by multiple alphaviruses while being completely permissive to flavivirus infection. The adaptive growth of a primary CHIKV strain through multiple passages allowed the emergence of a CHIKV strain that productively infected A549 cells while causing overt cytopathic effects and without a fitness cost for replication in otherwise CHIKV-susceptible cells. Whole genome sequencing of polyclonal and monoclonal preparations of the adapted virus showed that a limited number of mutations consistently emerged in both structural (2 mutations in E2) and non-structural proteins (1 mutation in nsP1 and 1 mutation in nsP2). The introduction of the adaptive mutations, individually or in combinations, into a wild-type molecular clone of CHIKV allowed us to determine the relative contributions of the mutations to the new phenotype. We found that the mutations in the E2 envelope protein and non-structural proteins contributed significantly to the acquired phenotype. The nsP mutations were introduced in a split-genome trans-replicase assay to monitor their effect on viral genome replication efficiency. Interestingly, neither mutation supported increased viral genomic replication in either Vero or A549 cells.