The Baltimore and Ohio Railroad was not really that significant on its own. However, it represents the beginning of the boom in American railroads, which was hugely significant.
When the B and O was first created, there was no good way of transporting bulky goods by land. Canals and rivers were the only feasible land means of transportation. This meant that it was very difficult to move goods from one place to another if there were no navigable waterways available. This severly limited the range of areas that were available for settlement and it placed limits on which areas could trade with one another.
The B and O represented a first step in changing this situation. It was started in 1828, reached a point 73 miles west of Baltimore 4 years later, but did not make it to the Ohio River until 1853. Meanwhile, railroads in general started to boom. The B and O did not really mean that much on its own, but it started the railroad industry in the US. Railroads in general played a huge role in allowing more parts of the country to be settled and creating a larger market for trade.