Difo made his Major League Baseball (MLB) debut in 2015 with the Washington Nationals and has also played for the Pittsburgh Pirates.

[6] On May 19, 2015, Difo was called up to the major leagues for the first time and collected his first hit in his first at-bat the same day, a pinch-hit single up the middle off New York Yankees reliever David Carpenter.

[7] Difo hit his first career home run on September 29, 2016, off Arizona Diamondbacks starter and former Nationals pitching prospect Robbie Ray.

[10] After Trea Turner suffered a broken wrist in a game days later, Difo took over as the Nationals' regular starting shortstop, occasionally sharing duties with Stephen Drew and Adrián Sánchez.

[11][12][13] On July 27, 2017, Difo hit one of four consecutive home runs by Nationals hitters off Milwaukee Brewers starter Michael Blazek, the first time the feat had been accomplished in Major League Baseball since the 2011 season.

[14] Splitting time between the Majors and Minors in 2019, Difo appeared in 43 games with the Nationals and had a .252 average with two home runs and eight RBIs.

Difo did not play in the postseason, but was on the Nationals' 40-man roster when the team won the 2019 World Series over the Houston Astros, their first championship in franchise history.

[18] On May 22, 2021, against the Atlanta Braves, Difo pitched one inning in a blowout, giving up eight runs on six hits, including a grand slam.

[28] During the 2017 season, Difo attracted some media attention for his choice of walk-up music, as he frequently had romantic ballads like "Unchained Melody" and "My Girl" added to his rotation.