Despite decades of research, the mechanism of action of the ABC multidrug transporter P-glycoprotein (P-gp) remains elusive. Due to experimental limitations, many researchers have turned to molecular dynamics simulation studies in order to investigate different aspects of P-gp function. However, such studies are challenging and caution is required when interpreting the results. P-gp is highly flexible and the time scale on which it can be simulated is limited. There is also uncertainty regarding the accuracy of the various crystal structures available, let alone the structure of the protein in a physiologically relevant environment. In this study, three alternative structural models of mouse P-gp (3G5U, 4KSB, 4M1M), all resolved to 3.8 Å, were used to initiate sets of simulations of P-gp in a membrane environment in order to determine: a) the sensitivity of the results to differences in the starting configuration; and b) the extent to which converged results could be expected on the times scales commonly simulated for this system. The simulations suggest that the arrangement of the nucleotide binding domains (NBDs) observed in the crystal structures is not stable in a membrane environment. In all simulations, the NBDs rapidly associated (within 10 ns) and changes within the transmembrane helices were observed. The secondary structure within the transmembrane domain was best preserved in the 4M1M model under the simulation conditions used. However, the extent to which replicate simulations diverged on a 100 to 200 ns timescale meant that it was not possible to draw definitive conclusions as to which structure overall was most stable, or to obtain converged and reliable results for any of the properties examined. The work brings into question the reliability of conclusions made in regard to the nature of specific interactions inferred from previous simulation studies on this system involving similar sampling times. It also highlights the need to demonstrate the statistical significance of any results obtained in simulations of large flexible proteins, especially where the initial structure is uncertain.