The Manchester City star - who netted 18 Premier League goals for the 2017-18 champions - teed up Ashley Young for another England chance in the 19th minute, but his close-range effort was blocked and smuggled to safety.
Sterling went in the book for taking an unnecessary tumble in the box and Eric Dier rose to head wide as England sought to reassert themselves.
The player sustained a hip injury and after undergoing three examinations - the last which took place in London on Thursday as the rest of the Super Eagles were preparing for a friendly against England - was ruled out of the quadrennial tournament on Friday.
While England were good in the first-half, Nigeria looked much better after the break.
Meanwhile, Lagos-born Iwobi and London-born Aina chose Nigeria for senior global football but they played alongside Alli for England's youth teams and it's this familiarity they're looking to tap into to put the Tottenham Hotspur star off his game at Wembley.
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"Now, we have our final 23 players for the World Cup".
20' FREEKICK! Young (England) is clipped by Onazi and the home side have another free-kick in a good position.
"I thought England played very well in the first-half against a poor Nigeria side", he told the BBC.
The 32-year-old said on ITV: "If you have the opportunity to play you can show what you do at club level, and I finished the season strongly so I am glad to be in the squad". But the visitors made their changes in the second-half and made a game of it. It was in the same manner of being alert and precise that Iwobi scored his two goals for Nigeria in the World Cup qualifiers, away and home to Zambia.
Southgate still has time to work on those tactical issues as England have one more friendly - against Costa Rica in Leeds on Thursday - before they fly to Russian Federation for their Group G opener against Tunisia in Volgograd on June 18.
I don't think we got a lot of answers though because Nigeria just lay down in the first period and let England do what they want.